Predicting Stricture in Morbidly Obese Patients Undergoing Laparoscopic Roux-en-Y Gastric Bypass : A Logistic Regression Analysis Gastrojejunostomy stricture after Roux-en-Y gastric bypass occurs in 3 to 27 % of morbidly obese patients in the USA . We questioned whether preoperative patient characteristics , including demographic attributes and comorbid disease , might be significant factors in the etiology of stricture . In this study from November 2001 to February 2006 -LRB- 51 months -RRB- , at a high-volume bariatric center , of the 1,351 patients who underwent laparoscopic gastric bypass , 92 developed stricture -LRB- 6.8 % -RRB- . All but two were treated successfully by endoscopic dilation . All patients stopped nonsteroidal anti-inflammatory medications 2 weeks prior to surgery and did not restart them . The operative procedure included the use of a 21-mm transoral circular stapler to create the gastrojejunostomy ; the Roux limb was brought retrogastric , retrocolic . In an effort to reduce our center 's stricture rate , late in the study , U-clips used at the gastrojejunostomy were replaced by absorbable sutures , and postoperative H2 antagonists were added to the treatment protocol . The change to absorbable polyglactin suture proved to be significant , resulting in a lower stricture rate . The addition of H2 antagonists showed no significant effect . Following the retrospective review of the prospective database , univariate and multivariate logistic regression analyses identified factors associated with the development of stricture . Gastroesophageal reflux disease and age were each shown to be statistically significant independent predictors of stricture following laparoscopic gastric bypass . Introduction Bariatric surgery , particularly Roux-en-Y gastric bypass -LRB- RYGB -RRB- , has been proven safe1 ,2 and effective in achieving long-term weight loss ,3,4 comorbidity reduction ,3,5 and enhancement of quality of life .6 Since its introduction by Wittgrove et al. in 1994,7 laparoscopic Roux-en-Y gastric bypass -LRB- LRYGB -RRB- has been shown to be as reliable as open RYGB8 -- 10 and less traumatic , requiring a briefer recovery time and shorter duration of stay .11 It is effective in achieving comparable weight loss at 1 year3 ,12 -- 15 and is successful in reducing the comorbidities of morbid obesity .3,9,13 Laparoscopic RYGB has surpassed other weight-loss methods for the morbidly obese in frequency of use in the USA .14 Laparoscopic RYGB is a technically demanding procedure with a lengthy learning curve of approximately 100 cases .16,17 Gastrojejunostomy stricture following RYGB has been reported variously in 3 to 27 % of patients .12,16 -- 19 Actual incidence of this complication is a function of the accuracy of its diagnosis and reporting19 and has been shown to be relative to operative technique and surgeon experience .20 Other known etiologies of stricture following LRYGB are tension on the anastomosis , foreign body reaction , technical error in creation of the anastomosis , marginal ulcer , and leak with associated scarring .19 In 2003 , Perugini and colleagues published an analysis of preoperative predictors of complications .20 They reported a 14.4 % rate of gastrojejunostomy stricture -LRB- N = 27/188 -RRB- , greater than half of all complications . With multivariate analysis by stepwise logistic regression , they found that surgeon experience was the most significant predictor of complications in general -LRB- not of stricture specifically -RRB- and that sleep apnea and hypertension were also significant independent predictors of complications .20 Perugini et al. cautioned that the factors they identified -LRB- experience , sleep apnea , hypertension -RRB- as predictive of complications in general might , in fact , `` be specific for the complication of gastrojejunal stenosis '' .20 Our study took up this question and , by univariate and multivariate logistic regression , analyzed preoperative predictors specifically for stricture . Patients and Methods Patients A consecutive series of morbidly obese patients underwent LRYGB between November 12 , 2001 , and February 28 , 2006 -LRB- 51 months -RRB- , in a dedicated , community-based , bariatric program awarded Center-of-Excellence status by the Surgical Review Corporation in 2005 . Institutional review board approval and informed consent were obtained prior to prospective data collection . In a retrospective analysis , preoperative patient characteristics , including whether the patient was taking any nonsteroidal anti-inflammatory medications on a chronic basis prior to surgery , were compared for two groups : those who developed gastrojejunostomy stricture -LRB- N = 17/92 , 6.8 % -RRB- and those who did not -LRB- N = 311/1 ,259 , 93.2 % -RRB- . No patients were excluded from the study . Patients where tested for Helicobacter pylori and treated if positive . Anti-inflammatory medications were withheld 2 weeks preoperatively and not resumed postoperatively . Procedures were performed by four bariatric surgeons . Stenosis , or stricture , indicated by progressive dysphagia , nausea , and vomiting , was diagnosed by endoscopy . Surgical Technique The procedure was performed using the transoral end-to-end anastomotic -LRB- EEA -RRB- technique similar to the Wittgrove technique .10 Using a Veress needle to an intra-abdominal pressure of 15 mm Hg , abdominal insufflation was performed . The patient was placed supine , in the reverse Trendelenburg position , and six abdominal trocars were introduced . The angle of His was dissected , and a 15-ml balloon was passed transorally and placed at the esophagogastric junction . The lesser sac was entered just below the balloon on the lesser curve of the stomach . A 15 - to 20-ml gastric pouch was created with the Endo GIA 45-mm stapler -LRB- Ethicon Endo-Surgery , Cincinnati , OH , USA -RRB- , using an average of three firings . A guidewire was introduced through the anterior abdominal wall and an endoscope was passed transorally . Cautery was used to pass the snare through the pouch and grasp the guidewire . The anvil of the 21-mm EEA Stealth stapler -LRB- Ethicon Endo-Surgery -RRB- was attached to the guidewire and brought down through the mouth . The ligament of Treitz was identified , and , approximately 20 cm distally , the jejunum was divided . A 100-cm Roux limb was created for patients with BMI < 55 kg/m2 , and a 150-cm Roux limb was created for patients with BMI ≥ 55 kg/m2 . The jejunojejunostomy was stapled side to side , and closed with the Endo GIA stapler . The Roux limb was brought retrocolic , retrogastric through the lesser curve of the stomach . The EEA circular stapler was introduced through the abdominal wall and the gastrojejunostomy was stapled end to end . The posterior wall of the gastrojejuostomy lays on soft tissue that holds the left gastric and vagus nerve and does not need reinforcement . All techniques for reinforcing the gastrojejunostomy were instituted only in the anterior wall of the anastamosis as a second layer . The initial technique was to sew a continuous second layer of the gastrojejunostomy anteriorly with 3 -- 0 polyglactin absorbable suture . The angle is often technically demanding . In October 2005 , the procedure was modified to one in which the gastrojejunostomy was oversewn with two Nitinol `` U '' clips that revert to preformed circular memory when placed , without additional suture . In October of 2005 , these U-clips were replaced with two interrupted 3 -- 0 polyglactin absorbable sutures . Finally , in January 2006 , the addition of H2 antagonists for 90 days to the postoperative regimen was made . These technical changes were instituted in an attempt to lessen the stricture rate , which is followed monthly in our database . Patients who experienced persistent postoperative nausea , vomiting , and intolerance to solid food were referred for upper gastrointestinal studies , followed by referral to the gastroenterologist for upper endoscopy . Patients with stricture were treated endoscopically with balloon dilation to a maximum of 15 mm . Statistical Methods Patient data were collected prospectively and included preoperative patient demographics and comorbidities , as well as postoperative complications extracted to identify potential influences on stricture rates . Variables included in the univariate logistic regression analysis are listed in Table 1 . Table 1Univariate Analysis to Identify Preoperative Patient Characteristics Associated with StrictureVariableStricture Group -LSB- N -LRB- % -RRB- -RSB- Nonstricture Group -LSB- N -LRB- % -RRB- -RSB- P ValueNo . patients -LRB- N = 1,351 -RRB- 921,259 Age , mean -LRB- SD -RRB- 41.4 -LRB- 11.6 -RRB- 44.4 -LRB- 10.4 -RRB- 0.039 aCardiac disease6 -LRB- 6.5 -RRB- 107 -LRB- 8.5 -RRB- 0.885 Chronic depression21 -LRB- 22.8 -RRB- 278 -LRB- 22.1 -RRB- 0.634 Chronic respiratory disease22 -LRB- 23.9 -RRB- 318 -LRB- 25.3 -RRB- 0.766 Chronic venous insufficiency53 -LRB- 57.6 -RRB- 692 -LRB- 55.0 -RRB- 0.347 Degenerative joint disease89 -LRB- 96.7 -RRB- 1,209 -LRB- 96.0 -RRB- 0.893 Diabetes , type 226 -LRB- 28.3 -RRB- 307 -LRB- 24.4 -RRB- 0.184 Ethnicity , N -LRB- % -RRB- -- -- 0.145 Caucasian76 -LRB- 82.6 -RRB- 1,122 -LRB- 89.1 -RRB- -- Other16 -LRB- 17.4 -RRB- 137 -LRB- 10.9 -RRB- -- GERD66 -LRB- 71.7 -RRB- 715 -LRB- 56.8 -RRB- 0.035 aSex , N -LRB- % -RRB- -- -- 0.208 Female81 -LRB- 88.0 -RRB- 1,042 -LRB- 82.8 -RRB- -- Male11 -LRB- 12.0 -RRB- 217 -LRB- 17.2 -RRB- -- Hypercholesterolemia/hyperlipidemia40 -LRB- 43.5 -RRB- 624 -LRB- 49.6 -RRB- 0.958 Hypertension41 -LRB- 44.6 -RRB- 652 -LRB- 51.8 -RRB- 0.838 Infertility11 -LRB- 12.0 -RRB- 96 -LRB- 7.6 -RRB- 0.732 Nonsteroidal anti-inflammatory medications17 -LRB- 18.5 -RRB- 294 -LRB- 23.4 -RRB- 0.284 No . preoperative comorbidities5 .2 -LRB- 2.0 -RRB- 5.1 -LRB- 1.9 -RRB- 0.259 No . preoperative medications4 .6 -LRB- 3.6 -RRB- 4.2 -LRB- 3.5 -RRB- 0.088 Obstructive sleep apnea43 -LRB- 46.7 -RRB- 564 -LRB- 44.8 -RRB- 0.415 BMI48 .5 -LRB- 8.5 -RRB- 49.2 -LRB- 8.3 -RRB- 0.202 Fasting blood sugar111 .5 -LRB- 45.4 -RRB- 108.9 -LRB- 39.6 -RRB- 0.779 HbA1C6 .2 -LRB- 1.8 -RRB- 6.1 -LRB- 1.2 -RRB- 0.150 Previous abdominal surgeries1 .2 -LRB- 1.1 -RRB- 1.2 -LRB- 1.3 -RRB- 0.754 Urinary stress incontinence52 -LRB- 56.5 -RRB- 698 -LRB- 55.4 -RRB- 0.322 aSignificance at the 0.05 level The SPSS ™ software package -LRB- version 14.0 , SPSS , Chicago , IL , USA -RRB- was used to perform all statistical analyses . Statistical significance was set at P < 0.05 . Binary logistic regression analysis was used in both univariate and multivariate modeling to identify independent preoperative variables associated with the development of gastrojejunostomy stricture following LRYGB surgery . Univariate analysis using logistic regression was applied to identify significant associations with the dichotomous outcome variable -LRB- stricture -RRB- . For comparison , Pearson chi-square test for categorical variables and Student 's t test for continuous variables were used . A priori preoperative factors of research interest -LRB- P < 0.21 -RRB- , as well as those found to be significantly associated with stricture -LRB- P < 0.05 -RRB- , were entered into multivariate analysis using forward stepwise logistic regression . Likelihood ratio tests were used for variable selection . In the initial analysis of preoperative characteristics by univariate logistic regression , age , number of comorbidities , number of medications , BMI , fasting blood sugar , HbA1C , and previous abdominal surgeries were classified as continuous variables . With the exception of sex and ethnicity -LRB- dichotomized to male/female and Caucasian/non-Caucasian , respectively -RRB- , all remaining variables were dichotomized -LRB- yes/no -RRB- . In the final multivariate forward stepwise logistic regression model , both age and BMI were redefined as categorical variables : age was coded to reflect four levels -LRB- ≤ 35 , 36 to 45 , 46 to 55 , and ≥ 55 years -RRB- and BMI was coded to reflect two levels -LRB- < 50 and ≥ 50 -RRB- . Results From November 12 , 2001 , to February 28 , 2006 -LRB- 51 months -RRB- , 1,351 patients underwent LRYGB at a single institution . Mean preoperative patient age was 44 years , mean BMI was 49 kg/m2 , 83 % of patients were female , and mean preoperative weight was 305 lb . Patients were predominantly Caucasian -LRB- 89 % -RRB- ; 7 % were Hispanic ; 3 % were African American ; and 1 % were of Native American , Asian/Pacific , and other ethnicities . Operative technique was the same in all procedures , namely , a retrocolic , retrogastric , transoral , 21-mm , circular stapled gastrojejunostomy . Mean intraoperative time was 96 min and length of stay averaged 2.8 days ; 97.6 % of operations were accomplished laparoscopically and 2.4 % were conversions to open procedures . There were two reoperations with revision of the gastrojejunostomy in the `` stricture group . '' The first was in a patient who developed a leak immediately postoperatively and returned to the OR for definitive management . The patient developed a stricture that was not amenable to dilation and , at 7 months , underwent a revision of her gastrojejunostomy . The second patient had a stricture that was dilated to 12 -- 15 mm . Subsequent endoscopy showed no recurrent stricture . She continued to eat very large portions in multiple meals , developing recurrent bezoars that had to be cleared endoscopically . The pouch dilated over time , and even though the gastrojejunostomy was shown to be only large enough to pass an endoscope , we were obliged to revise the pouch to a smaller size . Since that time she has had no further problem with bezoars . There were no deaths in the stricture group . In the `` nonstricture group , '' early mortality was 0.2 % -LRB- two deaths < 31 days postoperatively -RRB- ; intermediate mortality , 0.3 % -LRB- four deaths between 31 and 90 days postoperatively -RRB- ; and late mortality , 0.4 % -LRB- five deaths > 90 days postoperatively -RRB- . Ninety-two patients -LRB- 6.8 % -RRB- were diagnosed with stricture and underwent subsequent endoscopic dilation . Seventeen of 92 stricture patients -LRB- 18.5 % -RRB- were on nonsteroidal anti-inflammatory medications prior to surgery , whereas 294 of the 1,259 -LRB- 23.4 % -RRB- nonstricture patients were on nonsteroidal anti-inflammatory medications prior to surgery -LRB- Table 1 -RRB- . The difference in stricture rates between these two groups was not significant . Our rate of stricture -LRB- 92/1 ,351 , 6.8 % -RRB- was in the range of rates previously reported by Higa et al. -LRB- 5.3 % -RRB- , Schauer et al. -LRB- 4.7 % -RRB- , and DeMaria et al. -LRB- 6.6 % -RRB- , as compiled by Perugini20 , at the lower end of the national range of stricture prevalence . Other complications with the highest incidence were cholecystectomy -LRB- 6.5 % stricture group vs 3.0 % nonstricture group -RRB- , small bowel obstruction secondary to internal hernia -LRB- 4.4 vs 1.9 % -RRB- , and intra-abdominal abscess -LRB- 3.3 vs 2.0 % -RRB- . In the stricture and nonstricture groups , respectively , leakage occurred in 1.1 and 1.0 % of patients , and port site infection occurred in 2.2 and 3.6 % of patients . Peripheral neuropathy was 5.4 % in the stricture group and 0.5 % in the nonstricture group -LRB- Table 2 -RRB- . Table 2Forward Stepwise Multivariate Logistic Regression Model for Complications of StrictureVariableRate , N -LRB- % -RRB- P ValueOdds Ratio -LRB- 95 % Confidence Interval -RRB- Stricture GroupNonstricture Group30-day readmission27 -LRB- 29.35 % -RRB- 109 -LRB- 8.66 % -RRB- 0.000 a4 .381 -LRB- 2.666 -- 7.197 -RRB- Cholecystectomy6 -LRB- 6.52 % -RRB- 38 -LRB- 3.02 % -RRB- 0.181 NSInternal hernia1 -LRB- 1.09 % -RRB- 35 -LRB- 2.78 % -RRB- 0.337 NSSmall bowel obstruction4 -LRB- 4.35 % -RRB- 24 -LRB- 1.91 % -RRB- 0.500NSIntra-abdominal abscess3 -LRB- 3.26 % -RRB- 25 -LRB- 1.99 % -RRB- 0.691 NSPeripheral neuropathy5 -LRB- 5.43 % -RRB- 6 -LRB- 0.48 % -RRB- 0.000 a11 .979 -LRB- 3.423 -- 41.929 -RRB- Infection2 -LRB- 2.17 % -RRB- 2 -LRB- 3.57 % -RRB- 0.151 NSaSignificance at the 0.05 level Mean percent excess weight loss at 1 year following surgery was the same for both groups -LRB- − 103.1 lb , 82.6 % stricture group vs − 115.8 lb , 82.0 % nonstricture group -RRB- . Mean numbers of medications were reduced from 4.6 to 1.6 and from 4.2 to 1.5 in the stricture and nonstricture groups , respectively , at the most recent postoperative examination . By univariate logistic regression analysis , two preoperative characteristics were identified as factors associated significantly -LRB- P < 0.05 level -RRB- with stricture : gastroesophageal reflux disease -LRB- GERD -RRB- -LRB- P = 0.035 -RRB- and age -LRB- P = 0.039 -RRB- -LRB- Table 1 -RRB- . In addition to GERD and age , BMI -LRB- P = 0.202 -RRB- and gender -LRB- P = 0.208 -RRB- were incorporated into the final multivariate analysis . In the first multivariate model -LRB- Table 3 -RRB- , with age defined as a continuous variable , forward stepwise multivariate logistic regression confirmed GERD -LRB- P = 0.006 , OR = 1.917 , CI = 1.200 -- 3.062 -RRB- and age -LRB- P = 0.010 , OR = 0.973 , CI = 0.953 -- 0.993 -RRB- to be independent factors associated with stricture . Seventy-two percent of all stricture patients were diagnosed with GERD prior to surgery , and the mean age was 41 years , whereas , in the preoperative nonstricture group , 57 % were diagnosed with GERD and the mean age was 44 years . Table 3Significance and Odds Ratios for Age , GERD , Gender , and BMI in Multivariate Models to Predict StrictureVariableP ValueOdds Ratio -LRB- 95 % Confidence Interval -RRB- Multivariate model # 1a Age0 .010 b0 .973 -LRB- 0.953 -- 0.993 -RRB- GERD0 .006 b1 .917 -LRB- 1.200 -- 3.062 -RRB- Gender0 .324 NS Preoperative BMI0 .086 NSMultivariate model # 2c Age overall0 .015 Group 1 : ≤ 35 years -LRB- reference category -RRB- 1 Group 2 : 36 -- 45 years0 .0150.508 -LRB- 0.294 -- 0.877 -RRB- Group 3 : 46 -- 55 years0 .0040.431 -LRB- 0.244 -- 0.760 -RRB- Group 4 : > 55 years0 .1260.596 -LRB- 0.308 -- 1.156 -RRB- GERD0 .0051.963 -LRB- 1.227 -- 3.141 -RRB- Gender0 .353 NS Preoperative BMI0 .100 NSaAge classified as a continuous variablebSignificant at the 0.05 levelcAge classified as a categorical variable In the second multivariate model -LRB- Table 3 -RRB- , age was classified as a categorical variable to refine our understanding of the impact of age on stricture . Using age group 1 -LRB- ≤ 35 years -RRB- as the reference category , age groups 2 -LRB- 36 -- 45 years -RRB- and 3 -LRB- 46 -- 55 years -RRB- were significant at the 0.05 level -LSB- group 2 , P = 0.015 -LRB- OR = 0.508 , CI = 0.294 -- 0.877 -RRB- ; group 3 , P = 0.004 -LRB- OR = 0.431 , CI = 0.244 -- 0.760 -RRB- -RSB- . GERD remained significant -LRB- P = 0.005 ; OR = 1.963 , CI = 1.227 -- 3.141 -RRB- . Three postoperative complications were found to be associated significantly with the presence of stricture by univariate logistic regression , including readmissions within 30 days of surgery -LRB- P = 0.000 -RRB- , peripheral neuropathy -LRB- P = 0.000 -RRB- , and pneumonia -LRB- P = 0.031 -RRB- . The postoperative multivariate model determined readmissions within 30 days of surgery -LRB- P = 0.000 , OR = 4.381 , CI = 2.666 -- 7.197 -RRB- and peripheral neuropathy -LRB- P = 0.000 , OR = 11.979 , CI = 3.423 -- 41.929 -RRB- to be statistically significant covariates of stricture . Decreasing our rate of stricture by a protocol change to oversewing the gastrojejunostomy with polyglactin was shown , by chi square analysis , to be a significant decrease , with the rate of 6.8 % diminishing to 2.8 % -LRB- P = 0.028 , OR = 0.358 , CI = 0.143 -- 0.895 -RRB- . The second protocol adjustment , administration of an H2 antagonist -LRB- prevacid -RRB- postoperatively , had no effect . Discussion Strictures contribute to almost half of all readmissions in the early perioperative period and require instrumentation and rehydration . They can also be associated with the development of a more serious complication , peripheral neuropathy , as is demonstrated by these data . Specific intraoperative factors -LRB- e.g. , surgeon experience -RRB- associated with stricture have been identified as causal by prior studies . Isolating preoperative factors that may be causally related to stricture may aid in minimizing this complication . In their study , Perugini et al. showed preoperative hypertension and obstructive sleep apnea to be predictive of complications in general ; yet , they cautioned that their findings might have been confounded by their high rate of stricture .20 By logistic regression analysis , our study found the variables of GERD and age to be significantly associated with a complication with one of the highest incidences -- stricture . Our results are supportive of Perugini 's findings , in that we did not find the same preoperative predictors of complications -LRB- hypertension , obstructive sleep apnea -RRB- significantly associated with stricture and the overall rate was significantly less . The odds of developing stricture in patients with preoperative GERD were found to be nearly two times higher than those for patients without GERD . GERD is a known inflammatory condition of the upper gastrointestinal tract that specifically affects the lower esophagus and fundus . If present , this inflammation may predispose patients to more vigorous scarring of the gastrojejunostomy . During the period of the study , we began testing patients preoperatively for H. pylori . Patients who tested positive were treated preoperatively . In general , the micropouch used to create the gastrojejunostomy has been assumed to be almost devoid of acid-producing cells , and the rate of ulceration at the anastomosis at the time of endoscopy is low . It is clear , though , that adding H2 antagonists for 30 days following surgery helps to decrease inflammation , and in addition , the polyglactin stitch may be less inflammatory in nature . Data were insufficient at study summary to demonstrate the statistical significance of administering H2 antagonists ; however , we are encouraged by the trend of fewer strictures . Finding that younger rather than older age was associated with the development of stricture seems clinically counterintuitive . One reason for this is that age affects the small vessels ' ability to provide necessary oxygenation to the anastomosis , and older patients are more likely overall to have diminished capability to oxygenate . In our center , maintenance on oxygen the night of surgery is standard ; patients who require continuous positive airway pressure are started in the anesthesia recovery unit and are kept on supplemental oxygen until they can oxygenate in ambulating and resting modes at greater than 92 % . However , as stated , in our first multivariate model , older age was shown to be slightly protective with respect to stricture . In the second multivariate model , designed to refine the interpretation of age effects on stricture , we found that the odds of developing stricture in those 35 years or younger were two times greater than those aged 36 -- 45 years , and 2.32 times greater than those aged 46 -- 55 years . The trend of age being protective against stricture leveled off and was not significant in those older than 55 years . More study of this finding is indicated . Our original assumption that higher rates of central fat distribution in males might place greater tension on the anastomosis predisposing male patients , particularly those with higher BMIs , toward stricture , may be in error . The effect of increased central fat on the anastomosis may not be injurious if meticulous technique is used to ensure a tension-free anastomosis . Also , the ischemic effects of diabetes , sleep apnea , and chronic respiratory disease at the gastrojejunostomy site may be insufficient , in themselves , to cause stricture . It has been well documented that operative technique can contribute to increased stricture rates . Previous studies have reported fewer strictures with hand-sewn anastomoses than with stapled ones . Some authors have demonstrated that the 25-mm EEA stapler may be associated with fewer strictures that the 21-mm EEA stapler ; we have a 6.8 % stricture rate with the 21-mm EEA stapler . Further studies of stapling methodologies are warranted . It has been proposed that stricture rates may be lessened by the creation of a larger gastrojejunostomy anastomosis . The effect on the restrictive element of the bypass and subsequent effect on weight loss long term has not been evaluated . We avoided excessive dilation to allow the patient to maintain as much restriction as possible . Using a protocol of minimal dilation to between 12.5 and 15 mm resulted in only one perforation and acceptable weight loss . In our study and those of others ,21,22 weight loss at 1 year is not hindered by dilation . A limitation of this study is that is was retrospective . A prospective trial in which comorbidities were not controlled would not be feasible , although a prospective trial of H2 antagonists may be of value . It is difficult to discern the effect of absorbable suture on stricture rates vs the effect of H2 antagonists as their introduction to the protocol occurred in close proximity . The finding that readmissions within 30 days of surgery and postoperative peripheral neuropathy were statistically significant covariates of stricture most likely relates to the frequency of readmissions for peripheral neuropathy secondary to vitamin deficiencies following bariatric surgery . The objective of this study was to reach back in the causal continuum , prior to the intraoperative effects of technique and surgeon experience , to identify other potentially salient contributors to postoperative stricture . This analysis of a large series of patients undergoing RYGB identified GERD and age as factors associated with gastrojejunostomy stricture . Conclusions Using absorbable suture at the gastrojejunostomy anastomosis appears to decrease stricture rates . As identified via multivariate logistic regression analysis , GERD and age are independent predictors of gastrojejunostomy stricture .

O B O O O O O O O B I O O O O O B B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O B I O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B B O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O B B O

obesity
bariatric surgery
stricture
gastric bypass
gastrojejunostomy


Altered spin state equilibrium in the T309V mutant of cytochrome P450 2D6 : a spectroscopic and computational study Cytochrome P450 2D6 -LRB- CYP2D6 -RRB- is one of the most important cytochromes P450 in humans . Resonance Raman data from the T309V mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzyme 's active site perturbs the heme spin equilibrium in favor of the six-coordinated low-spin species . A mechanistic hypothesis is introduced to explain the experimental observations , and its compatibility with the available structural and spectroscopic data is tested using quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V mutant . Introduction Cytochromes P450 -LRB- CYPs -RRB- are enzymes responsible for the oxidative biotransformation of a large variety of endogenous and exogenous substrates , like steroids , carcinogens and drugs , and have been found in virtually all organisms -LSB- 1 -RSB- . In human drug metabolism , one of the most important CYP isoforms is CYP2D6 . This hepatic enzyme is involved in the phase I oxidative metabolism of about 30 % of currently marketed drugs , including much prescribed antidepressants , β-blockers , opioids and antiarythmics -LSB- 2 -RSB- . The enzyme is known for its genetic polymorphisms and gene multiplicities , even increasing its clinical relevance -LSB- 3 -RSB- . Structural studies on CYP2D6 are of crucial importance to understand how this enzyme works and to be able to predict the metabolism of new -LRB- druglike -RRB- compounds -LSB- 4 -RSB- . A common structural feature in all CYPs , including CYP2D6 -LSB- 5 -RSB- , is the distal helix -LRB- I-helix -RRB- positioned above the heme plane , which constitutes an important part of the enzyme 's active site -LRB- Fig. 1a -RRB- . Previously , we demonstrated the critical role of the I-helix residue T309 in the mechanism of oxidation by CYP2D6 -LSB- 6 -RSB- . Similar results were found after mutation of the homologous threonine residues T302 and T303 in rabbit CYP2B4 and CYP2E1 , respectively -LSB- 7 , 8 -RSB- . The I-helix threonine is highly conserved in CYPs and seems to be a key residue in the monooxygenation reaction . It has been studied in many CYPs with mutagenesis , spectroscopic and crystallographic techniques -LSB- 9 -RSB- : depending on the specific isoenzyme , the distal threonine has been attributed a variety of roles besides oxygen activation and proton delivery -LSB- 9 , 10 -RSB- , including electron transfer -LSB- 11 -RSB- , substrate recognition -LSB- 12 -RSB- and control of heme spin state equilibrium -LSB- 12 -- 17 -RSB- . This multiplicity of roles , as indicated by the diversity of results obtained from different CYPs , has been interpreted as a lack of a universal role of this residue , which might have instead distinct functions within the CYP superfamily -LSB- 9 , 14 , 17 -RSB- . Fig. 1a The cytochrome P450 2D6 -LRB- CYP2D6 -RRB- active site as obtained from crystallographic data -LRB- PDB 2F9Q -RRB- . The heme -LRB- coordinated by C443 -RRB- and part of the I-helix are shown in color with the heme and the residues A305 and T309 as ball-and-stick models . Hydrogen atoms are not modeled in the PDB file . b Optimized geometry of the CYP2D6 active site model , i.e. , the A305 -- G306 -- M307 -- V308 -- T309 part of the I-helix and the iron porphyrin part of the heme catalytic center of CYP2D6 in its doublet -LRB- low-spin -RRB- electronic spin state , with a water molecule coordinated to the iron -LRB- position 1 -RRB- . c Geometry of the CYP2D6 active-site model corresponding to a second energy minimum -LRB- water in position 2 -RRB- . Carbon , nitrogen , oxygen and hydrogen atoms are colored green , blue , red and white , respectively . Iron and sulfur atoms are colored orange . Distances are in angstroms In this study , spectroscopy is used to probe the active site of the wild type and the T309V mutant of CYP2D6 to establish whether the mutation influences the heme characteristics . Resonance Raman -LRB- RR -RRB- , a vibrational spectroscopy technique based on inelastic scattering of laser radiation in resonance with an electronic transition of a chromophore , is used to obtain information about several properties of the heme prosthetic group -LSB- 18 , 19 -RSB- . The heme-iron oxidation state , spin state and coordination state , as well as conformational changes of the vinyl and propionate side chains of the porphyrin macrocycle can be inferred from RR spectra -LSB- 19 -RSB- . Furthermore , the Fe -- CO stretching frequency of the ferrous CO -- heme complex is sensitive to variations in the electrostatic potential of the heme distal pocket induced by substrate-binding or mutated residues -LSB- 20 , 21 -RSB- . RR was successfully employed in the past to study many CYPs -LSB- 19 -RSB- and the effects of mutations in their active site -LSB- 22 -- 27 -RSB- , including those of the conserved I-helix threonine -LSB- 11 , 28 , 29 -RSB- . Besides spectroscopic data , a computational approach is employed to rationalize the experimental results . Although experimental data on threonine mutants are already available for many CYPs , theoretical descriptions have been only carried out at a classical force-field level for the T302A mutant of CYP2B4 -LSB- 30 -RSB- and at a density functional theory -LRB- DFT -RRB- level for the T252A mutant of CYP101 -LSB- 31 -RSB- . Quantum-mechanical DFT calculations can reliably predict the relative energies of the various electronic states of iron -LRB- II -RRB- and iron -LRB- III -RRB- porphyrins and have been previously used to study the resting state as well as further steps of the catalytic cycle of CYPs -LSB- 31 -- 35 -RSB- . Therefore , this method , in absence of decisive experimental proof in favor of a specific model , is used in the present work to test the compatibility of a definite hypothesis with available structural and spectroscopic data on the CYP2D6 active site . In particular , through a computational analysis of the wild-type and the T309V-mutant active-site structures , interactions are investigated between residue T309 , the heme moiety and a water molecule located above the heme in the resting state . Materials and methods Materials The pSP19T7LT plasmid containing in tandem the complementary DNAs -LRB- cDNAs -RRB- of human CYP2D6 with a C-terminal His6-tag and the human NADPH-CYP reductase , was used as described before -LSB- 6 -RSB- . Escherichia coli JM109 was obtained from DSMZ -LRB- Braunschweig , Germany -RRB- . Dextromethorphan hydrobromide -LRB- DX -RRB- was obtained from Sigma -LRB- St. Louis , MO , USA -RRB- . All other chemicals were of analytical grade and were obtained from standard suppliers . Expression and purification of the enzymes The pSP19T7LT plasmids containing the wild type or the T309V mutant of CYP2D6 cDNA were transformed into E. coli strain JM109 . Expression and membrane isolation was carried out as described in -LSB- 6 -RSB- . Membranes were resuspended in 0.5 % of the original culture volume of potassium phosphate -- glycerol buffer -LRB- 50 mM potassium phosphate buffer , pH 7.4 , 10 % glycerol -RRB- and the enzymes were purified , using nickel affinity chromatography as described in -LSB- 6 -RSB- . CYP concentrations were determined by CO difference absorption spectra according to the procedure of Omura and Sato -LSB- 36 -RSB- , with a Pharmacia Ultrospec 2000 spectrometer . RR spectroscopy Spectroscopic measurements were conducted using a homebuilt Raman microscope in a backscattering configuration . A Zeiss microscope -LRB- D-7082 with × 40 objective , numerical aperture 0.60 , working distance 2 mm -RRB- was coupled to a single monochromator -LRB- Instruments , Metuchen , NJ , USA -RRB- with a mounted grating with 2,400 grooves per millimeter and a CCD camera -LRB- DV-420OE , Andor Technologies , Belfast , UK -RRB- . The 413.1-nm line of a continuous-wave Kr ion laser -LRB- Coherent Innova 300c , Coherent , Santa Clara , CA , USA -RRB- and the 457.9-nm line of a continuous-wave Ar ion laser -LRB- Spectra Physics 2000 -- 336 , Newport Corp. , Mountain View , CA , USA -RRB- were used for excitation , and the Rayleigh scattered light was removed using third Millennium edge long pass filters -LRB- Omega Optical , Bratleboro , VT , USA -RRB- . Laser powers of 5 mW -LRB- at 413.1 nm -RRB- and 1 mW -LRB- at 457.9 nm -RRB- at the sample were used throughout the experiments . The sloping background of the spectra was subtracted using a baseline fitted to the experimental data with the Andor CCD camera software . The fitting of experimental data with Lorentzian functions was performed with PeakFit 4.12 -LRB- SeaSolve Software , Richmond , CA , USA -RRB- . For RR measurements , a 1-mm-diameter glass capillary was filled with 10 μl of various concentrations -LRB- 1 -- 50 μM -RRB- of CYP2D6 in potassium phosphate -- glycerol buffer and put in a spinning capillary holder under the microscope objective . To measure substrate binding , substrate concentrations of 5 -- 10 mM were used , leading to a 500 -- 1,000-fold excess of substrate with respect to the enzyme -LRB- saturating conditions , as indicated by the Ks reported for the substrates -LSB- 6 -RSB- -RRB- . No further spectral changes were detected upon increasing substrate concentration , indicating that all the CYP2D6 in the sample was bound to the substrate -LSB- 37 -RSB- . To obtain the ferrous CO complexes , oxygen was removed from the CYP2D6 samples -LRB- with or without substrate -RRB- by stirring under a nitrogen atmosphere ; then the samples were reduced by adding 5 μl of 25 g l − 1 sodium dithionite -LRB- in potassium phosphate -- glycerol buffer -RRB- to 20 μl of enzyme in the same buffer , and were exposed to CO for 2 -- 3 min while stirring . During all measurements , the capillary containing the sample was kept spinning to minimize local heating and photodissociation of the iron-bound CO. . Before and after every RR measurement , CYP integrity was monitored by CO difference absorption spectroscopy . Computational details Model The crystal structure at 3.0-Å resolution of the substrate-free human CYP2D6 -LRB- PDB 2F9Q -RRB- -LSB- 5 -RSB- was used as the starting structure for the DFT calculations . The computational model includes only a small part of the distal I-helix from A305 to T309 , the heme moiety and the proximal C443 . In this model , the isopropyl amino acid side chain of V308 is replaced by a methyl substituent and the 2 - -LRB- methylthio -RRB- ethyl side chain of M307 is replaced by a hydrogen . These alterations are applied to considerably reduce the computational cost and are usually justified by the fact that these amino acid side chains are not pointing toward the heme cavity -LSB- 38 -RSB- . Likewise , the heme substituents are replaced by hydrogens , and the C443 proximal heme ligand is mimicked by a thiolate anion -LRB- HS − -RRB- . Moreover , a water molecule is added above the heme iron to generate a six-coordinated -LRB- 6c -RRB- resting state , as suggested by spectroscopic data -LRB- see `` Results '' and `` Discussion '' -RRB- . This results in an 89-atom model for the active site of the wild-type protein . The T309V mutant is approximated by replacing the alcohol moiety of the T309 by a methyl substituent to give V309 instead . These two models are used to provide a rationale for the differences between the observed relative amounts of high spin -LRB- HS -RRB- and low spin -LRB- LS -RRB- in the wild-type and T309V-mutant enzymes of CYP2D6 . Method DFT calculations were performed with the Amsterdam Density Functional program -LSB- 39 -- 41 -RSB- . The atomic orbitals on the atoms of the alcohol moiety of T309 , the carbonyl group of A305 , the thiolate anion , iron and nitrogens of the heme moiety were described by an uncontracted triple zeta valence plus polarization -LRB- TZP -RRB- Slater-type orbital -LRB- STO -RRB- basis set . A double zeta valence plus polarization -LRB- DZP -RRB- STO basis set was used for the other atoms of the I-helix and the atoms of the periphery of the heme . The inner cores of carbon , nitrogen , and oxygen -LRB- 1s2 -RRB- and those of sulfur and iron -LRB- 1s22s22p6 -RRB- were kept frozen . The exchange -- correlation potential was based on the newly developed generalized gradient approximation exchange functional OPTX in combination with the nonempirical PBE correlation functional -LRB- OPBE -RRB- -LSB- 42 , 43 -RSB- . The OPBE density functional was chosen because of its demonstrated superior performance in describing the spin states and electronic structures for iron complexes , which is of crucial importance in the present study -LSB- 32 -RSB- . The model was based on the geometry of 2F9Q -LSB- 5 -RSB- and is fully optimized for the doublet spin state -LRB- LS -RRB- . This structure was used as a starting structure for the linear transit calculations -LRB- geometry scan -RRB- in which the water molecule was moved toward the threonine by decreasing the Owater -- OThr distance to yield the five-coordinated -LRB- 5c -RRB- heme moiety . In these geometry optimizations , the positions of the α carbons of A305 and T309 and two porphyrin meso carbons were fixed to mimic the rigidity of the protein tertiary structure -LRB- see supplementary material -RRB- . Subsequently , analogous geometry optimizations of the LS geometries obtained were performed for the sextet spin states -LRB- HS -RRB- . The energy profiles for the mutant enzyme model were obtained by the same procedure for each structure of the wild-type profile in which the threonine hydroxyl moiety was replaced by a methyl substituent . Results Enzymes expression and purification , UV -- vis absorption spectroscopy Recombinant expression followed by affinity chromatography purification yielded approximately 60 nmol CYP2D6 per liter of cultured E. coli , in the case of the wild type and the T309V mutant of CYP2D6 . The enzymes were pure , as judged by the observation of a single band of 55 kD on a Coomassie brilliant blue stained sodium dodecyl sulfate -- polyacrylamide gel . The absolute absorbance spectra of oxidized and reduced CO-bound , wild-type and T309V-mutant CYP2D6 are shown in Fig. 2 . Both enzymes show characteristic strong absorption bands at 418 and 448 nm , in their resting state and reduced CO-bound form , respectively . No significant differences are observed in the position or the relative intensity of absorption bands between the spectra of the wild type and the T309V mutant of CYP2D6 , even by use of spectral subtraction -LRB- data not shown -RRB- . An amount of P420 -LRB- inactive CYP , observed as a band at 420 nm in the reduced CO-bound form -RRB- was present in the both the wild-type and T309V-mutant samples , to approximately the same extent . Fig. 2Electronic absorption spectra of 1 μM purified wild-type and T309V-mutant CYP2D6 , in the oxidized resting state form -LRB- solid line -RRB- and the reduced CO-bound form -LRB- dashed line -RRB- RR spectroscopy The RR spectra of the wild type and the T309V mutant of CYP2D6 in the resting state -LRB- i.e. , without substrate -RRB- are at first sight rather similar -LRB- Fig. 3 -RRB- . In both cases , the most intense band at 1,372 cm − 1 is attributed to the ν4 vibrational mode , indicative for an oxidized heme iron atom -LSB- 19 -RSB- . Fig. 3Resonance Raman spectra of wild-type -LRB- WT -RRB- and mutant -LRB- T309V -RRB- CYP2D6 . Spectra were acquired as described in `` Methods . '' Excitation wavelength 413.1 nm , laser power at the sample 5 mW , accumulation time 600 s The high-frequency region of the RR spectrum of the wild-type enzyme -LRB- Fig. 4 -RRB- is indicative for a predominantly 6cLS oxidized heme , in agreement with a previous study -LSB- 27 -RSB- . The bands at 1,501 , 1,582 and 1,635 cm − 1 are attributed to the ν3 , ν2 and ν10 vibrational modes for a 6cLS heme , respectively -LSB- 19 -RSB- . However , the slight asymmetry of ν3 suggests the presence of another band at lower wavenumbers , characteristic of a 5cHS species . A better fit of the spectroscopic data for wild-type CYP2D6 is obtained when including bands at 1,486 , 1,567 and 1,623 cm − 1 for , respectively , the ν3 , ν2 and ν10 vibrational modes of a 5cHS heme species in addition to the 6cLS bands . Fig. 4Spin marker band region of the resonance Raman spectra of wild-type -LRB- WT -RRB- and mutant -LRB- T309V -RRB- CYP2D6 , together with their difference spectrum . Excitation wavelength 413.1 nm , laser power at the sample 5 mW , accumulation time 600 s . The Lorentzian functions used to fit the spectrum of the wild-type enzyme -LRB- see `` Methods '' -RRB- are represented as solid lines -LRB- for high-spin marker bands -RRB- or dotted lines -LRB- low-spin marker bands -RRB- . Frequencies of high-spin marker bands are reported in bold The presence of 5cHS heme species in the RR spectrum of the wild type is more evident when compared with the spectrum of the T309V mutant -LRB- Fig. 4 -RRB- . Although the two spectra seem similar , the difference spectrum of the two enzymes shows an intensity increase for the bands characteristic of a 6cLS species and a concomitant decrease of the ν3 , ν2 and ν10 vibrational modes that are typical for a 5cHS heme . Therefore , in the T309V mutant the content of 5cHS is decreased compared with that in the wild type , with a concomitant increase of the 6cLS fraction . In the presence of saturating amounts of the substrate DX , the bands distinctive of the 5cHS species -LRB- ν3 and ν2 at 1,486 and 1,567 cm − 1 , respectively -RRB- increase in intensity , whereas the intensity of the corresponding bands for the 6cLS species -LRB- ν3 and ν2 at 1,501 and 1,582 cm − 1 , respectively -RRB- diminishes for both the wild type and the T309V mutant of CYP2D6 -LRB- Fig. 5 -RRB- . The intensity ratio between the 6cLS and 5cHS ν3 bands -LRB- at 1,501 and 1,486 cm − 1 , respectively -RRB- is higher in the T309V mutant than in the wild type for substrate-bound CYP2D6 . Fig. 5Spin marker bands region of the resonance Raman spectra of wild-type -LRB- WT -RRB- and mutant -LRB- T309V -RRB- CYP2D6 in the presence -LRB- solid lines -RRB- and in the absence -LRB- dotted lines -RRB- of the substrate dextromethorphan hydrobromide . The spectral difference between the enzyme in the presence and in the absence of substrate is shown for the spin marker band ν3 . Excitation wavelength 413.1 nm , laser power at the sample 5 mW , accumulation time 600 s The T309V mutation does not alter the Fe -- CO stretching frequency -LRB- νFe -- CO -RRB- observed at 476 cm − 1 in both the reduced CO-bound wild-type and the T309V-mutant CYP2D6 -LRB- Fig. 6 -RRB- . As previously observed -LSB- 27 -RSB- , in the RR spectrum of wild-type CYP2D6 νFe -- CO increases by 5 cm − 1 upon addition of saturating amounts of DX . In the same experimental conditions , the νFe -- CO band of the T309V mutant broadens toward higher wavenumbers , indicating that a shift , although less evident , is taking place similarly to that in the wild type . Fig. 6Low-frequency resonance Raman spectra of the reduced CO-bound CYP2D6 wild type -LRB- WT -RRB- and mutant -LRB- T309V -RRB- , in presence and in absence of dextromethorphan hydrobromide -LRB- DX -RRB- . Excitation 457.9 nm , laser power at the sample 1 mW , accumulation time 600 s DFT calculations The optimized geometry of the enzyme 's resting state was modeled by adding a water molecule above the heme using the crystal structure of the human CYP2D6 -LRB- Fig. 1b -RRB- . The root mean square deviation of the optimized backbone of the I-helix and the corresponding residues in the experimental structure is only 0.28 Å , confirming that the modifications applied to the side chains of residues M307 and V308 in the model do influence the conformation of the I-helix in the active site . The water molecule is bound to the iron -LRB- water position 1 in Fig. 1b , Fe -- O distance is approximately 2.2 Å -RRB- , yielding a 6cLS iron porphyrin complex that is energetically favored over the 6cHS state by 7.5 kcal mol − 1 . The water molecule is hydrogen-bonded to the carbonyl oxygen atom of the A305-G306 peptide bond -LRB- approximately 1.9 Å -RRB- . The hydroxyl moiety of the threonine amino acid residue -LRB- T309 -RRB- also forms a weak hydrogen bond to this amide oxygen -LRB- approximately 2.6 Å -RRB- . When the water molecule migrates from the iron to the threonine moiety , a hydrogen bond is formed -LRB- Fig. 1c -RRB- in which the threonine alcohol group acts as a hydrogen-bond donor ; the conformation with the OH group acting as a hydrogen-bond acceptor is 6.6 kcal mol − 1 less stable . The energy profiles upon migration of the water molecule toward the threonine -LRB- i.e. , from position 1 to 2 -RRB- for both the LS and the HS states are shown as solid curves in Fig. 7 . This movement of the water molecule results in the increase of the relative energy of the LS state and a new local minimum -LRB- 5cLS -RRB- is obtained , which is 1.4 kcal mol − 1 higher in energy with respect to the energy of the starting structure -LRB- 6cLS -RRB- . The relative energy of the HS state increases slightly upon shortening the threonine -- water distance and decreases again to reach a new minimum that is lower in energy . There is a spin crossover at a Owater -- OThr distance of approximately 3.1 Å and a global minimum for the HS state is obtained . In the corresponding structure , the water molecule is hydrogen-bonded to the T309 and occupies a second position in the active-site pocket , further referred to in the following sections as position 2 -LRB- Fig. 1c -RRB- . Fig. 7The relative doublet -LRB- low spin , filled circles -RRB- and sextet -LRB- high spin , filled squares -RRB- energies for the active-site model of the wild-type CYP2D6 with respect to the distance between the oxygen atoms of the water molecule and the threonine alcohol side group . The dashed line represents the low-spin -LRB- open circles -RRB- and high-spin -LRB- open squares -RRB- energy profiles for the mutant model , obtained by replacing threonine with valine . LS low spin , HS high spin , 5c five-coordinated , 6c six-coordinated Replacement of the threonine with valine in each of the structures followed by partial geometry optimizations results in different energy profiles for the mutant -LRB- dashed curves in Fig. 7 -RRB- . For both the HS and the LS mutant models , after a small initial decrease of approximately 2 kcal mol − 1 which yields minima at an Owater -- OThr distance of 3.9 and 3.5 Å for the LS and HS states , respectively , the relative energy increases steeply as the water approaches position 2 , for which , in contrast to the wild type , no minima are found . The interaction between residue T309 and the CO adduct was studied by full geometry optimization of the CO-bound iron porphyrin model species for both the wild type and the T309V mutant , showing that the bond distances , bond dissociation energies and bond orders of the Fe -- C ≡ O moiety are virtually identical -LRB- Table 1 -RRB- . Population analysis of the CO adduct shows a substantial iron backdonation into the empty π * orbitals of CO , with occupation numbers of approximately 0.2 electrons for both the wild type and the mutant . Table 1Selected bond distances , bond dissociation energies -LSB- ΔE -LRB- Fe -- CO -RRB- -RSB- , bond orders and π * -LRB- CO -RRB- orbital population of the CO -- heme model for the wild type and the T309V mutant of cytochrome P450 2D6Fe -- COFe -- C ≡ OWild typeMutantWild typeMutantDistance -LRB- Å -RRB- 1.8241.8511.1471.146 ΔE -LRB- Fe -- CO -RRB- -LRB- kcal mol − 1 -RRB- − 24.6 − 24.2 -- -- Mayer bond order0 .210.200.520.52 π * -LRB- CO -RRB- population -- -- 0.19 e − 0.18 e − The superimposed geometries of the fully optimized and experimental structures , of the partially optimized structures of the T309V mutant with water in positions 1 and 2 , and of the fully optimized structures of the CO-bound species for both the wild type and the T309V mutant are available as supplementary material . Discussion In the resting state , the CYP heme iron atom is generally observed as oxidized -LRB- Fe3 + -RRB- , and mainly in the 6cLS state with a water molecule -LRB- or hydroxyl ion -RRB- as the sixth distal ligand trans to the endogenous cysteinate ligand -LSB- 44 -RSB- . Although no distal water ligand is reported in the structure determined by X-ray diffraction -LSB- 5 -RSB- -LRB- Fig. 1a -RRB- , both electronic absorption and RR spectroscopy indicate that wild-type CYP2D6 is predominantly 6cLS . The present DFT calculations show that the resting state is clearly a 6cLS state , in agreement with spectroscopic data -LRB- Fig. 1b -RRB- . In addition to experimental data , DFT calculations suggest a network of hydrogen bonds between the residue A305 , the distal water ligand and the residue T309 , in which T309 and the water ligand are too far to interact directly . The same structural pattern , where the distal water is hydrogen-bonded to an alanine -LRB- four positions before the conserved threonine in the primary sequence -RRB- , which is in turn weakly hydrogen bonded to the threonine , is also present in the experimental structures of other bacterial and mammalian CYPs -LSB- 45 -- 47 -RSB- . Moreover , in agreement with the DFT calculations for CYP2D6 , in all these structures the conserved threonine is too far to hydrogen-bond the distal water ligand . The absence of a direct interaction between the heme axial ligand and the threonine in CYP2D6 is experimentally corroborated by the absence of differences in the electronic absorption and RR spectra of the CO adducts of the wild type and T309V , where a CO ligand occupies the position of the water molecule in the resting state . In fact , hydrogen bonding or direct electrostatic interaction between T309 and the axial CO would alter the extent of backdonation of iron dπ electrons to the empty CO π * orbitals , shifting significantly both the Fe -- CO stretching frequency in RR spectra and the absorption maximum in the UV -- vis absorption spectra of the reduced CO-bound heme -LSB- 48 , 49 -RSB- . A similar situation is observed for the T268A mutant of CYP102 A1 , whose CO adduct has a UV -- vis absorption spectrum identical to that of the wild type -LSB- 9 , 14 -RSB- . The absence of a significant interaction between the CO adduct and residue T309 is supported by DFT calculations on CO-bound CYP2D6 , showing that the Fe -- CO bond energy , bond order , and the extent of Fe → CO backdonation are virtually the same for both the wild type and the mutant -LRB- Table 1 -RRB- . Besides the predominant 6cLS state , the 5cHS species is present as a minor component in RR spectra of CYP2D6 -LRB- Fig. 4 -RRB- . The occurrence of the 5cHS species could not be detected in UV -- vis absorption spectra , probably because of its small amount . RR spectra have more , narrower bands which are usually better resolved than UV -- vis spectra at room temperature , making it easier to detect asymmetries or weak shoulders when using spectral subtraction or fitting . The presence of the 5cHS species in the resting state is not unusual and it has been reported in RR spectra of other bacterial and mammalian CYPs -LSB- 50 -- 53 -RSB- . A shift of the spin equilibrium toward the 5cHS state , in which the heme is no longer ligated to an axial water molecule , is often observed when a substrate -LRB- like DX for CYP2D6 ; Fig. 5 -RRB- is present in the heme pocket , perturbing the iron-bound water molecule directly , or through inducing conformational changes in the enzyme -LSB- 44 -RSB- . However , this substrate-induced spin shift often does not lead to a pure 5cHS species , but rather to a mixture of the two species , with 6cLS still significantly present , like in the case of CYP2D6 with DX . The decrease of 5cHS and the concomitant increase of the 6cLS content in the RR spectra of the T309V mutant of CYP2D6 indicate that the residue T309 is structurally involved in the spin equilibrium . This role is preserved in the presence of DX as the substrate itself perturbs the wild-type and mutant spin equilibria to the same extent , which is evident from the spectral subtraction of the ν3 region of the resting state from the substrate-bound state -LRB- Fig. 5 -RRB- . The same conclusion can be drawn from RR spectra of the CO adduct of the two enzymes in the presence of substrate . Moreover , the similar affinity of the two enzymes for DX -LRB- as was judged from optical titration experiments -LSB- 6 -RSB- as well as modeling studies -LSB- 54 -RSB- -RRB- does not indicate T309 to be involved in substrate binding in CYP2D6 . This strongly suggests that the difference in the 6cLS-to-5cHS ratio in the RR spectra between the substrate-bound wild-type and mutant enzyme is not due to different binding modes of DX , but rather to a difference caused by the T309V mutation , as observed in the absence of a substrate . An altered spin equilibrium upon mutation of the conserved threonine residue has been reported for many other CYPs -LSB- 12 , 14 -- 17 , 55 , 56 -RSB- . Notably in the T268A mutant of CYP102 A1 , which share with the optimized CYP2D6 computational model the hydrogen-bonding pattern `` distal water -- alanine -- threonine , '' the alteration is in favor of the 6cLS , like in CYP2D6 -LSB- 12 , 14 -RSB- . In an effort to rationalize the experimental observations , we looked for an explanatory model , compatible with our spectroscopic data , which could be tested using available quantum-mechanical methods . The absence of direct interactions between T309 and the axial ligand indicates that T309 exerts its influence on the spin equilibria of CYP2D6 indirectly . It might be argued that the increase of the 6cLS species in the T309V mutant is due to an increased solvent accessibility of the binding pocket caused by a mutation-induced conformational change . However , this hypothesis is incompatible with the spectroscopic data on the CO adduct , which rule out any significant change in polarity in the heme pocket due to the T309V mutation . A more plausible explanation is that T309 in CYP2D6 stabilizes , by means of hydrogen bonding , an alternative position of the water that is distant enough from the iron center to yield a 5cHS state . This model has also been proposed to account for spin equilibria in substrate-bound CYP102 A1 and its T268A mutant -LSB- 13 , 14 -RSB- . Substitution of the threonine with a valine should eliminate the hydrogen-bonding interaction and perturb the equilibrium by destabilizing the 5cHS state in favor of the 6cLS species , as observed from spectroscopic data . DFT calculations clearly support this hypothesis , showing the destabilization of the 5cHS state upon the T309V mutation . In agreement with the experimental observation of a small amount of 5cHS in the resting state of the wild type , a spin transition from LS to HS occurs upon moving the water molecule from position 1 to 2 , and a second energy minimum has been found for the water molecule hydrogen-bonded to T309 -LRB- Figs. 1c , 7 -RRB- . Replacing the threonine with a valine results in a relative destabilization of the structure in which water is in position 2 , most likely because of the absence of hydrophilic interactions between the water and the valine . This relative destabilization of the 5c heme with the water in position 2 will result in the occurrence of a higher relative amount of the 6cLS heme species in the T309V mutant of CYP2D6 with respect to the wild type . Although the computational model of the CYP2D6 active site used in the present study only includes a few amino acids and the iron -- porphyrin complex , and therefore does not take into account the rest of the protein , it provides a clear rationale for the observed experimental data . Unfortunately , an evident limit of this model is the inability to include the substrate and to study its influence on the spin equilibrium of the substrate-bound enzymes , for which extended models and methods have to be used . However , it should be stressed that experimental data indicate that the role of T309 in the CYP2D6 spin equilibrium is independent of that of DX ; therefore , it is reasonable to assume that the implications of our model for the role of T309 would still be valid for the substrate-bound enzymes . Conclusions On the basis of RR data , it is concluded that the T309V mutant of CYP2D6 has an altered spin equilibrium with respect to the wild-type enzyme , with a relative higher amount of 6cLS species at the expense of the 5cHS species , in both the resting state and the substrate-bound forms . Apparently , there is no direct interaction between residue T309 and the heme sixth ligand , suggesting an indirect mechanism of action on the spin equilibrium . Spectroscopic data also indicate that the T309V mutation does not significantly alter the polarity of the heme environment , excluding an increased number of water molecules in the heme pocket as the reason for the altered spin state . DFT calculations show that a simple model , involving a water molecule alternatively occupying two positions inside the heme pocket corresponding to two different spin states , is able to explain the experimental data . In this model , the position corresponding to the 5cHS state is stabilized by a hydrogen bond with T309 , and a T309V mutation will induce an increase of the 6cLS species , as experimentally observed . Electronic supplementary material Below is the link to the electronic supplementary material . Supplementary material -LRB- PDF 215 kb -RRB-

O O O O O O B O O B I I O O O O O O B I I O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B I O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

cytochrome p450 2d6
resonance raman
t309v
spin equilibrium


Experiments Suggest that Simulations May Overestimate Electrostatic Contributions to the Mechanical Stability of a Fibronectin Type III Domain Steered molecular dynamics simulations have previously been used to investigate the mechanical properties of the extracellular matrix protein fibronectin . The simulations suggest that the mechanical stability of the tenth type III domain from fibronectin -LRB- FNfn10 -RRB- is largely determined by a number of critical hydrogen bonds in the peripheral strands . Interestingly , the simulations predict that lowering the pH from 7 to ∼ 4.7 will increase the mechanical stability of FNfn10 significantly -LRB- by ∼ 33 % -RRB- due to the protonation of a few key acidic residues in the A and B strands . To test this simulation prediction , we used single-molecule atomic force microscopy -LRB- AFM -RRB- to investigate the mechanical stability of FNfn10 at neutral pH and at lower pH where these key residues have been shown to be protonated . Our AFM experimental results show no difference in the mechanical stability of FNfn10 at these different pH values . These results suggest that some simulations may overestimate the role played by electrostatic interactions in determining the mechanical stability of proteins . Fibronectin is an extracellular matrix protein composed of three types of repeating domains -LRB- type I , type II and type III -RRB- . The type III -LRB- fnIII -RRB- domains -LRB- which are ubiquitous in many multidomain mechanical proteins -RRB- in particular have been found to play a pivotal role in regulating and mediating physiological functions of cells . This is achieved through the interaction of various domains with the integrin family of cell surface receptors .1 One of the critical interactions in fibronectin is the binding of the RGD -LRB- Arg-Gly-Asp -RRB- motif in the tenth fnIII domain of fibronectin -LRB- FNfn10 -RRB- to cell-surface integrins . A linear RGD-peptide has been shown to bind -LRB- with reduced binding strength and selectivity -RRB- to different members of the integrin family .2 It may be the dynamic structure of these domains that determines the functional states of the protein . Indeed , it has been suggested that force-induced conformational change of these fnIII domains may be crucial in transmitting cellular signals .3 Thus knowledge of how fnIII domains respond to mechanical forces and their mechanical resistance to conformational change is of importance to understand the function of fibronectin at the molecular level . A number of studies have investigated the mechanical properties of the fnIII domains of fibronectin by both experiment4 ,5 and simulation .6 -- 8 Using steered molecular dynamics -LRB- SMD -RRB- simulations , Vogel and co-workers6 predicted a `` mechanical hierarchy '' of a number of fnIII domains that is in reasonable qualitative agreement with the hierarchy obtained from the atomic force microscopy -LRB- AFM -RRB- forced unfolding experiments .4 One of the most mechanically weak fnIII domains is the tenth fnIII domain of human fibronectin , FNfn10 , although , interestingly , this is thermodynamically the most stable fnIII domain to have been studied to date .9,10 The simulations suggested that the mechanical stability of FNfn10 is largely determined by the hydrogen bonds between the A and B strands around Arg6 and Asp23 -LRB- Figure 1 -RRB- . Recent solution studies by Koide and co-workers10 showed that FNfn10 becomes more thermodynamically stable at lower pH -LRB- < 5.0 -RRB- as a consequence of the protonation of three negatively charged residues : Asp7 , Asp23 and Glu9 , which have raised pKa values of 5.54 , 5.40 and 5.25 , respectively ; they are essentially fully protonated below pH 4.7 . Using their simulations , Vogel and co-workers6 showed that protonation of these side-chains allows them to move closer together to form side-chain -- side-chain hydrogen bonds . They suggested that this stabilizes interactions between the A and B strands , resulting in a significant increase in the unfolding force in simulations at pH 4.7 even though it is well established that there is no correlation between mechanical stability and thermodynamic stability .11,12 This interesting result suggests that the mechanical stability of fibronectin might be modulated by a change in the pH of the tissues . We tested this prediction by monitoring the unfolding force of FNfn10 at different pH values : pH 4.5 -LRB- 50 mM acetate -RRB- , pH 5.0 -LRB- 50 mM acetate -RRB- and pH 7.0 -LRB- 50 mM phosphate -RRB- . It is important to note that Asp7 , Asp23 and Glu9 are fully protonated at pH 4.5 as shown by NMR experiments carried out by Koide and co-workers.10 Any increase in mechanical stability due to the protonation of these three residues , as shown in the SMD simulation , should be observed in the AFM experiments . Forced unfolding experiments were performed for a polyprotein containing eight repeats of the FNfn10 domain . Figure 2 shows a `` typical '' force-extension trace . As has previously been observed5 Fnfn10 unfolds either by a two-state unfolding mechanism -LRB- N → U , where N is the native state and U the unfolded state -RRB- or a three-state unfolding mechanism via an intermediate -LRB- I -RRB- -LRB- N → I → U -RRB- . The unfolding forces for three transitions could thus be determined : FN → U , the force of unfolding of N directly to U ; FN → I , the force of unfolding of N to I ; and FI → U , the force of unfolding of I to U -LRB- Figure 2 -RRB- . The intermediate , I , has been shown , using site-directed mutagenesis , to be a species with the A-strand detached .5 Thus one might expect , if the simulations of the unfolding of FNfn10 are correct , that both the N → U and N → I transitions will be at higher force at lower pH due to the new stabilising interactions between the A and B strands . However , since the A strand is detached in I , any stabilizing interactions between the A and B strands should not affect the mechanical stability of the intermediate , and FI → U is expected to be unaffected by pH. Furthermore , the unfolding forces of N → U are higher than those of N → I. Li et al. 5 suggested that this may be due to the `` stochastic nature '' of mechanical unfolding . They suggested that since N → I unfolding is observed at higher unfolding forces than the subsequent I → U unfolding , the I → U unfolding may sometimes occur at a force that is too low to be seen in the AFM force-extension traces .5 That is , the unfolding is likely always to occur via this intermediate but it may not be observed , particularly when N unfolds at high forces . Thus if the simulations are correct we might expect to see fewer I → U transitions at lower pH. To our surprise , the unfolding forces at all pH values are the same within error -LRB- Figure 3 -RRB- . This is true at all pulling speeds . The dependence of the unfolding force on the pulling speed remains unchanged , suggesting that there has been no change in the unfolding pathway . Note also that the proportion of I → U unfolding events remains about the same . Thus our results are in direct contradiction to the predictions from the steered molecular dynamics simulations . AFM combined with protein engineering Ф-value analysis and molecular dynamics simulations has been previously used to solve the mechanical unfolding pathway of TNfn3 , a homologous fnIII domain from the extracellular matrix protein tenascin .13 The results suggest that the unfolding of fnIII domains is a complicated multi-step process . The major barrier to a forced unfolding event in TNfn3 is the conformational transition from a twisted to an aligned state , which involves the breaking of several key hydrogen bonds along the peripheral strands -LRB- A-B strands and some between the G and F-strands -RRB- . But there is also significant loss of hydrophobic side-chain packing interactions of residues in the A , B and G-strands , and importantly there is significant core re-packing . Thus , hydrophobic contacts of the buried residues and the hydrogen bonding interactions along peripheral strands are both apparently critical to the mechanical stability of TNfn3 . TNfn3 and FNfn10 fold into essentially identical tertiary structures encompassing seven beta-strands running in two antiparallel beta sheets . A structural alignment of TNfn3 and FNfn10 reveals that these two fnIII domains have essentially the same hydrogen bonding patterns14 and molecular dynamics simulations have suggested that they have similar forced unfolding pathways . It is therefore reasonable to anticipate that these two fnIII domains will have the same molecular determinants for mechanical stability . We have made a `` core-swap '' version of FNfn10 , with the core of Tnfn3 which is significantly more stable than Fnfn10 itself .15 Considering these together , it seems that the mechanical stability of both FNfn10 and TNfn3 is likely to be associated with the complex interplay between key peripheral hydrogen bonds and hydrophobic effects from the packing of buried residues . Molecular dynamics simulations have previously proved to be of great value in predicting and understanding the behavior of proteins placed under mechanical stress .6,7,11,13,16,17 However , our results suggest that in the case of the simulations of forced unfolding of FNfn10 tested here ,6 the relative strength and importance of electrostatic and hydrophobic components may not be adequately described . It is possible that this discrepancy lies in the different timescale of the simulations and the experiments . AFM experiments are typically performed at extension rates of ∼ 1 μm s − 1 , whereas the timescale of molecular dymamics simulations allows for full extension of the protein in 1 or 2 ns , equivalent to a pulling speed of several m s − 1 , many orders of magnitude faster . Many slow conformational changes in proteins will not be observed in simulations on the nanoseconds timescale . Furthermore , the pathway of forced unfolding may vary as a function of the loading rate .18,19 Our study serves to emphasize the point that simulations need constantly to be benchmarked against experiment . However , the fact that simulators are prepared to make ab initio predictions that can be tested experimentally can only serve to improve our understanding of molecular mechanisms underlying mechanical strength in proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

afm
forced unfolding
extracellular matrix

fnfn10, the tenth fniii domain of human fibronectin

tnfn3, the third fniii domain of human tenascin