among natural amino 
 acids histidine is one of the strongest metal 
 ion coordinators .
hence , histidine plays 
 an important role in metal ion coordination in proteins and peptides . 

histidine is a tridentate ligand , which provides ligands at the imidazole 
 imido nitrogen , the amino nitrogen , and the carboxylate oxygen .
the imidazole ring nitrogen of histidine residues 
 often provides the primary coordination site for metal ions .
copper is an essential metal ion for biological 
 functions and is found in bound forms in metalloproteins and in low 
 molecular weight complexes to avoid toxicity associated with free 
 copper .
copper - containing proteins often 
 have binding sites with irregular geometries containing one or more 
 histidine ligands .
copper coordination in amyloidogenic proteins 
 such as amyloid- 
 ( a ) , prion , and -synuclein is achieved through histidine 
 residues .
small changes in the 
 coordination may have an effect on aggregation and other chemical 
 mechanisms in the diseased state .
hence , 
 it is critical to elucidate the structural details of the different 
 coordination modes to completely understand the biological role played 
 by metal  protein complexes . in -synuclein two
cu(ii ) coordination sites at the n - terminal 
 region were identified using mass spectrometry ( ms ) and nuclear magnetic resonance ( nmr ) .
later circular dichroism ( cd ) and electron spin resonance ( esr ) results indicated that these sites 
 are independent of each other .
the high affinity site is anchored 
 by the n - terminus , and the second site is anchored by the histidine 
 at position 50 .
other than these two 
 sites , recent nmr and esr results propose a low affinity cu(ii ) binding 
 site at the c - terminal region .
in prion protein the 
 presence of four binding sites at higher cu(ii ) occupancy in each 
 of the phgggwgq octarepeat regions were identified based on esr , cd , nmr , and x - ray absorption spectroscopy 
 ( xas ) results .
two additional cu(ii ) equivalents 
 coordinate via the histidine residues located at positions 96 and 
 111 .
additionally , the cu(ii ) coordination environment 
 in prion octarepeat domain has been extensively studied by ab initio 
 methods .
the copper coordination to a is highly 
 heterogeneous , with 
 the specific coordination environment depending on the ph , ionic strength , 
 concentration of the metal ions , and the presence of other metal ions . 

the metal binding domain of a contains three histidine residues 
 at positions 6 , 13 , and 14 .
the presence of all 
 three histidine residues in cu(ii ) coordination spheres was initially 
 proposed by nmr and was later confirmed by esr .
other techniques such as xas , fourier transform infrared spectroscopy ( ftir ) , cd , ultraviolet 
visible ( uv  vis ) , ms , and ab initio 
 calculations also proposed the involvement of histidine residues in cu(ii ) coordination . 
 a number of esr studies have suggested an equilibrium between two 
 different cu(ii ) coordination modes in an equimolar acu(ii ) 
 complex at ph 7.4 , known as component i and component ii .
component ii is dominant at higher ph ( ph 
 8.7 ) . these two components are believed 
 to have a different number of histidine residues coordinated to the 
 cu(ii ) ion .
component i is believed to 
 have two histidine residues that simultaneously coordinate to cu(ii ) .
previously we used electron spin echo envelope modulation ( eseem ) 
 in conjunction with n isotopic labeling , to determine 
 that the a component i consists of three subcomponents , at 
 physiological ph where two of the three histidines are simultaneously 
 coordinated to cu(ii ) ion .
interestingly , 
 only two of these subcomponents , are present at lower ph values .
cu(ii ) coordinated to his 6his 13 and his 6his 14 
 are found in equal proportions , while the his 13his 14 coordination 
 is absent at lower ph .
these measurements were able 
 to uncover the critical role of his 13his 14 in cu(ii ) coordination 
 at physiological ph and provide a possible rationale for the presence 
 of amorphous aggregates , rather than fibrils at high cu(ii ) concentrations .
the number of histidine residues coordinated to cu(ii ) in 
 component 
 ii is controversial .
one hypothesis proposes that cu(ii ) is coordinated 
 to the co group of the ala 2glu 3 bond and three histidine 
 residues .
the second proposition involves 
 the n - terminus , one n atom from one of the three histidine imidazoles , 
 the backbone n from asp 1-ala 2 peptide bond , and the co group of 
 the ala 2glu 3 peptide bond .
the 
 coordination of cu(ii ) in component ii is believed to be highly dynamic . 

the elucidation of the coordination in component ii is critical as 
 a recent research shows that , in the presence of zn(ii ) , cu(ii ) coordination 
 moves to a component ii - like coordination .
furthermore , it has been shown that zn(ii ) can only displace cu(ii ) 
 from component i coordination .
one equivalent 
 of zn(ii ) ions displaces 25% of the bound cu(ii ) from a(116 ) 
 at physiological ph , while rearranging the rest of the bound cu(ii ) 
 in component i. in the presence of one 
 equivalent of zn(ii ) ions , components i and ii account equally for 
 cu(ii ) coordination in a. however , 
 at excess amounts of zn(ii ) , component ii becomes the dominant cu(ii ) 
 coordination mode . in brain tissues affected 
 by the alzheimer s disease
, the concentration of zn(ii ) is 
 approximately three times higher than cu(ii ) .
therefore , component ii cu(ii ) coordination may be the most dominant 
 coordination mode in vivo .
shedding light into the coordination of 
 component ii is critical to understand the behavior of cu(ii ) in ad 
 etiology .
herein , we propose a method to quantify the number 
 of n nuclei coordinated to a cu(ii ) center by the use 
 of integrated 
 intensities of the fourier - transformed eseem .
eseem spectroscopy is 
 a pulsed esr technique that has been used to identify and quantify 
 the number of histidine imidazoles coupled to a cu(ii ) center .
spectral 
 simulations of the eseem spectra are used to determine the number 
 of coupled n nuclei .
compared the normalized integrated intensities of the 
 frequency domain eseem spectra to calculate the intensity reduction 
 resulting from the replacement of a n with n in a(116)cu(ii ) complexes .
although , the use of integration method was useful in elucidating 
 important structural information , the validity of the method was not 
 tested experimentally .
here we obtained eseem data from simple model 
 complexes with different numbers of imidazoles coordinated to cu(ii ) . 

the normalized integrated intensities of the model complexes increased 
 monotonically , when the number of imidazole rings increased in model 
 complexes .
two small peptides with known cu(ii ) coordination was used 
 to test the validity of the method . the dahk 
finally , our method was used to distinguish between the two proposed 
 modes of cu(ii ) coordination . then , in conjunction with n isotopic labeling we quantified the subcomponent proportions in 
 component ii .
for 
 the preparation of tetrakisimidazolecopper(ii ) sulfate ( see figure 1a ) , 40 ml of a 1 m imidazole ( 99% sigma - aldrich 
 co. , st .
louis , mo ) solution was added to 10 ml of 1 m cuso45h2o ( 98+% sigma - aldrich co. , st .
after a few days , dark 
 blue colored crystals formed . for the 
 preparation of bis(2-methylimidazole)copper(ii ) diacetate ( see figure 1b ) , 0.5 
 g of cu(ii ) acetate hydrate ( 98% sigma - aldrich co. , st .
louis , mo ) 
 and 1.0 g of 2-methylimidazole ( 99% sigma - aldrich co. , st .
louis , 
 mo ) were added and dissolved in a mixture of chloroform ( 10 ml ) and 
 methanol ( 2.5 ml ) .
then , 15 ml of diethyl ether was added to the filtrate 
 and stirred for 10 min .
then another 5 ml of diethyl ether was added 
 and filtered under reduced pressure and washed with diethyl ether 
 and chloroform .
the solid was air - dried and recrystallized from methanol / diethyl 
 ether . for dienimidazolecopper(ii ) diperchlorate ( see figure 1c ) , 30 ml of 2 mm cu(clo4)26h2o ( 98% acros organics , new jersey ) in a methanol / acetonitrile 
 ( 5:1 ) mixture was stirred with 3 ml of 2 mm triethylenediamine ( 97% 
 fluka , netherlands ) in water .
then , 10 ml of 2 mm imidazole in methanol 
 was added to the mixture . after being left overnight ,
all the solvents 
 used in synthesis were purchased from sigma - aldrich co. , st .
crystals were dissolved in water to make 10 mm stock solutions . 
 for esr experiments ,
samples were prepared in the presence of 25% 
 ( v / v ) glycerol ( 99% sigma - aldrich co. , st .
louis , mo ) , with 
 a final cu(ii ) concentration of 1.25 mm .
structures 
 of the model complexes with 1 , 2 , and 4 imidazole rings 
 and their crystal structures .
the peptide phgggw , the cu(ii ) binding domain of prion , was 
 synthesized 
 at the molecular medicine institute , university of pittsburgh , using 
 standard fluorenylmethoxycarbonyl chemistry .
isotopically enriched [ g - n]-n-fmoc - n-trityl - l - histidine was purchased from cambridge 
 isotope laboratory ( andover , ma ) , in which all nitrogen atoms are n enriched .
three different variants of amyloid-(116 ) 
 ( daefrhdsgyevhhqk ) with each containing an n enriched 
 histidine at either position 6 , 13 , or 14 were synthesized at the 
 molecular medicine institute , university of pittsburgh , using standard 
 fluorenylmethoxycarbonyl chemistry .
double - labeled 
 peptides containing two n enriched histidine residues 
 were synthesized in the same manner .
all the labeled amyloid- 
 ( 116 ) variants were purified by high - performance liquid chromatography 
 and characterized by mass spectroscopy .
nonlabeled amyloid- 
 ( 116 ) peptide was purchased from rpeptide ( bogart , ga ) .
the 
 peptide fragment dahk , the n - terminus region of the human serum albumin , 
 was purchased from fisher scientific , hanover park , il .
( 116 ) peptide was purchased from rpeptide ( bograt , ga ) . for 
 peptide samples ,
the concentration of the peptide was 1.25 mm and 
 an equimolar amount of cu(ii ) was present in both the samples .
samples 
 were prepared in n - ethylmorpholine ( nem ) buffer at 
 ph 7.4 in 25% ( v / v ) glycerol and appropriate amounts of hydrochloric 
 acid .
x - ray diffraction 
 data for the one - imidazole and two - imidazole model complex structures 
 were collected using a single crystal on a bruker x8 prospector ultra 
 ccd diffractometer with a cuk (  = 1.54178 a ) imus radiation 
 source .
the parameters used during the collection of diffraction data 
 for each structure are summarized in supporting 
 information .
crystals were mounted in fluorolube oil on a mitegen 
 mount and placed in a cold n2 stream ( 150(1 ) k ) for data 
 collection .
x - ray diffraction data for the four - imidazole model complex 
 was collected on a bruker smart apex ccd diffractometer with graphite - monochromated 
 mok (  = 0.71073  ) radiation at room temperature . 

for each structure , unit - cell dimensions were derived from the least - squares 
 fit of the angular settings of 999 strong reflections from the data 
 collection .
each structure was solved via direct methods , which 
 located the positions of the non - hydrogen atoms .
an inspection 
 of fo vs fc values and trends based upon sin  , miller index , or parity 
 group failed to reveal any systematic error in the data .
all computer 
 programs used in the data collection and refinements are contained 
 in the bruker program package apex2 v.2013.100 .
all 
 three crystal structures have been reported previously , and 
 our results are in substantial agreement with those previously reported 
 structures .
a 200 l 
 aliquot of complex samples with a concentration of 1.25 mm was transferred 
 into a quartz tube with an inner diameter of 3 mm .
all esr experiments were performed 
 on a bruker elexsys e580 x - band or a bruker elexsys e680 x - band ft / cw 
 spectrometer equipped with bruker er4118x - md5 and en4118x - md4 resonators , 
 respectively .
the temperature was controlled using an oxford itc503 
 temperature controller and an oxford cf935 dynamic continuous flow 
 cryostat connected to an oxford llt 650 low - loss transfer tube .
continuous - wave 
 esr experiments were carried out on sample solutions at 80 k and at 
 x - band microwave frequency .
the magnetic field was swept from 2600 
 to 3600 g for 1024 data points .
a time constant of 10.24 ms , a conversion 
 time of 20.48 ms , modulation amplitude of 4 g , a modulation frequency 
 of 100 khz , and a microwave power of 0.1993 mw were the other instrument 
 parameters used for the cw experiment .
the three - pulse eseem 
 experiments were performed on the sample solutions at 20 or 80 k , 
 by using a /2    /2  t  /2-echo pulse sequence with a /2 
 pulse width of 16 ns .
the first pulse separation ,  , was set 
 at 144 ns , and the second pulse separation , t , was 
 varied from 288 ns with a step size of 16 ns .
the experiments were 
 carried out at the magnetic field , where the esr intensity was maximum . 

after the baseline correction , the spectra were fast fourier - transformed . 
 then the final spectra were obtained as the magnitude of the fourier 
 transforms .
we normalized 
 the eseem spectra 
 using the integrated intensity of h eseem signal ( 1316 
 mhz ) .
possible limitations of the above method are discussed in the results section . for a single n nucleus coupled to an electron spin , the relative modulation depth 
 is1where k is the 
 modulation 
 depth and subscripts 14 and 1 denote the n and h spin , respectively .
superscripts  and  denote the 
  and  spin manifolds of the electron spin , respectively . for two equivalent n nuclei 

coupled to an electron 
 spin the relative modulation depth becomes2 k14 is the modulation depth of the  spin 
 manifolds of the two equivalent n nuclei . the relative 
 modulation depth of n increases with the addition of 
 the n nucleus , and the factor of increase is given by3 if k14 and k14 are much 
 smaller than 
 1 , the factor converges to 2 .
the theoretical value for k14 and k14 is approximately 0.15 , for a /2 pulse length of 16 ns .
if two nonequivalent n nuclei are coupled to the electron 
 spin , the relative modulation depth is given as4k14 is the modulation depth 
 of the  spin manifolds of the two nonequivalent n nuclei . the increase in the relative modulation depth with the 
 additional n nuclei
is given by5if k14 , k14 , k14 , and k14 are much smaller than 1 , eq 5 simplifies to6if 
 two n nuclei are equivalent 
 ( i.e. , k14 = k14 ) , the factor in 
 eq 6 becomes 2 .
however , obtaining modulation 
 depth information from eseem time 
 domain data is difficult as many components are overlaid in the signal . 

it has been shown that integrated intensities can be used to account 
 for the changes in the modulation depth . in the frequency domain n eseem signal
is well separated 
 from the h eseem signal , so it is possible to integrate 
 separately .
the n eseem intensity is obtained by integrating 
 between 011 mhz , and the h eseem intensity is 
 obtained for the region between 1316 mhz .
in this work we experimentally determine the validity of the use 
 of eseem integration intensities to quantify the number of n nuclei coupled to a cu(ii ) center . to this end a series of model 
 complexes with different numbers of imidazole ligands coordinated 
 to copper are used .
then , the eseem analysis was carried out on two 
 small peptides with known cu(ii)histidine coordination , namely 
 dahk and phgggw peptides .
the dahk peptide is the n - terminus region 
 of the human serum albumin ( hsa ) and coordinate cu(ii ) in the well 
 characterized atcun motive . the dahk 
cu(ii ) 
 complex contains a single n eseem active nucleus through 
 the histidine imidazole coordination .
the peptide phgggw is a truncated 
 fragment of the octarepeat cu(ii ) binding domain of the prion protein 
 and has a known cu(ii ) coordination including a crystal structure .
cu(ii ) complex is specifically 
 used as there are two nonidentical n eseem active nuclei 
 coupled to cu(ii ) .
one n nucleus is from the histidine 
 imidazole coordination and the other from the peptide backbone coordination .
then we used eseem to distinguish between the 
 two proposed modes of coordination for component ii of a. furthermore , 
 the proportions of each histidine residue coordinated to cu(ii ) ion 
 as an equatorial ligand were measured with the use of systematic n - labeled histidine residues . in copper 
imidazole 
 complexes , the imidazole ring has a noncoordinated distal nitrogen , 
 which contributes toward the eseem signal at x - band frequencies ( 9.5 
 ghz ) , with pulse lengths used in this work ( /2 = 16 ns ) . hence , the n eseem intensity will 
 increase with the addition of imidazole rings to the coordination . 
 in order to quantify the increase in n eseem intensity 
 we synthesized three model complexes ( shown in figure 1 ) containing 1 , 2 , and 4 imidazoles coordinated to a copper 
 center , respectively .
all three complexes were synthesized as crystals , 
 and the structure of the complexes was verified using x - ray crystallography . 

all these structures contain copper centers coordinated 
 with four equatorial ligands according to the information gained from 
 crystal structures . in the one - imidazole and the four - imidazole complexes , 
 structures of the counterion ligands perchlorate and the sulfate , 

esr measurements are less sensitive to axial ligands and are not considered in the analysis .
the 
 one - imidazole complex contains four directly coordinated nitrogens ; 
 three from the tridentate ligand , and one from the imidazole ring . 

directly coordinated nitrogens do not contribute to eseem at x - band , 
 with pulse lengths used in this work .
the one - imidazole model complex 
 contains just single n eseem active nuclei , which is 
 in the imidazole ring .
as shown in figure 2 , the one - imidazole complex has g and a values of ( 2.22  
 0.005 ) and ( 191  1 ) gauss , respectively .
these esr parameters 
 correspond to four nitrogen nuclei coordinated to the cu(ii ) center 
 in the equatorial plane , which is consistent 
 with the structure of the one - imidazole complex , as shown in figure 1a .
the two - imidazole complex has g and a values 
 of ( 2.30  0.005 ) and ( 158  1 ) gauss , respectively , which 
 is consistent with a two nitrogen and two oxygen nuclei equatorial 
 coordination .
for the four - imidazole 
 complex , g and a values are ( 2.26  0.005 ) and ( 182  
 1 ) gauss , respectively , which again is consistent for four directly 
 coordinated nitrogen nuclei .
hence , cw - esr 
 parameters clearly show that model complexes maintain the same cu(ii ) 
 coordination environment in solution .
nuclear quadrupole interactions ( nqi ) of n 
 give rise to features below 2 mhz . the broad feature around 4 mhz 

is due to the double quantum ( dq ) transition of the remote nitrogen 
 in an imidazole ring .
the intensity of the dq peak 
 increases with the number of imidazole rings coordinated to the cu(ii ) 
 center .
a peak around 9 mhz ( black arrow 
 in figure 3 ) is also indicative of multiple 
 imidazole coordination .
this peak is clearly 
 observed in the two- and four - imidazole complexes . experimentally obtained 
 three - pulse eseem spectra of the model 
 complexes at the maximum g position . 

appearance of a peak around 9 mhz in two- and four - imidazole complexes 
 is indicative of multiple imidazole coordination .
the integrated intensity for n - eseem was calculated 
 for the region between 011 mhz and then divided by the h - eseem intensity integrated between 13 and 16 mhz .
details 
 of the error calculation are provided in the supporting 
 information ( see figure s3 ) .
the normalized n - eseem 
 intensity increases from 8.4 to 21 when going from single imidazole 
 to two imidazoles as shown in table 1 . when 
 there are four imidazoles coordinated to the cu(ii ) center , normalized n - eseem intensity is increased to 40 .
hence , there is a monotonic 
 increase in the normalized n - eseem intensity with the 
 increase of number of imidazole rings coordinated . in the text ,
values in the last 
 column are referred as normalized integrated intensity . in order to test our claim in a biologically 
 relevant system ,
eseem experiments were conducted on two different 
 peptide fragments with well - known cu(ii ) coordination .
the four amino 
 acid peptide dahk is the n - terminus fragment of the human serum albumin . in the dahk peptide
, the imidazole ring of the 
 histidine residue coordinates to the cu(ii ) ion , and the distal nitrogen 
 of the imidazole ring is the only eseem active nuclei as shown in 
 figure 4 .
the comparison between the eseem 
 spectra of cu(ii)dahk and one - imidazole complex is shown in 
 figure 4 .
the intensity of dq peak reflects 
 the number of imidazoles coordinated to the cu(ii ) center .
the normalized integrated intensity for cu(ii)dahk 
 is 7.0  0.1 compared to 8.4  0.1 for the one - imidazole 
 complex ( table 1 ) .
only one n - eseem active nuclei is present in 
 the reported coordination of dahk .
then we used the phgggw peptide fragment , which is the cu(ii ) 
 binding 
 domain of the prion protein .
resolved 
 the cu(ii ) coordination environment of the octarepeat fragment using 
 esr experiments and a crystal structure of the cu(ii)phgggw 
 complex .
as shown in figure 5 , cu(ii ) is coordinated to an imidazole nitrogen 
 in a histidine , two backbone nitrogens from two glycine residues , 
 and an oxygen from a carboxylic group .
the structure of the cu(ii ) 
 bound hgggw pentapeptide was found to be unstable in solution due 
 to the breaking of the axial water coordination
also , eseem results 
 have indicated that histidine imidazole coordination and backbone 
 coordination is present in the cu(ii)phgggw complex in solution .
the eseem spectra of the cu(ii)phgggw complex and the two - imidazole 
 complex are shown in figure 5 for comparison . 

given the different coordination environments the two spectra do not 
 have identical peak positions .
the two - imidazole complex has a larger dq peak 
 compared to the phgggw complex .
the reported structure for the cu(ii)phgggw 
 structure contains two n - eseem active nuclei from the 
 distal nitrogen of the imidazole histidine and the backbone coordination 
 as shown in figure 5 .
hence , the intensity of dq peak is expected to be different between 
 the two complexes , as dq peak intensity is indicative of the number 
 of imidazoles coordinated .
however , the normalized n - eseem integrated intensity obtained for the cu(ii)phgggw 
 complex is similar to the integrated intensity of the two - imidazole 
 complex .
this information verifies that the integration method can 
 predict the number of n nuclei coupled to a cu(ii ) center .
comparison 
 of eseem spectra of cu(ii)phgggw complex and 
 the two - imidazole complex .
the integration 
 analysis was used to determine the number of nitrogens coupled to 
 the cu(ii ) ion in a(116 ) in component ii .
as shown 
 in the inset of figure 6 , zn(ii ) selectively 
 displaces cu(ii ) coordinated to component i. at excess amounts of 
 zn(ii ) ( ten equivalents of zn(ii ) ) , component ii accounts for 65% 
 of the overall coordination ( inset of figure 6 ) , where in the absence of zn(ii ) the percentage is only 35% .
amyloid aggregates in brain tissues contain 
 approximately three times zn(ii ) than cu(ii ) .
figure 6 shows the comparison between 
 the two - imidazole complex and the a(116)cu(ii ) 
 complex at ph 8.7 . at ph 8.7 only the component ii of cu(ii ) coordination 
 exists .
the integrated intensities tabulated in 
 table 1 suggest that two n nucleus 
 are coupled to the cu(ii ) ion .
the features of the eseem spectrum 
 clearly illustrate the imidazole histidine coordination as shown in 
 figure 6 .
the backbone 
 coordination peak is possibly due to the coupling between the cu(ii ) 
 ion with the remote backbone nitrogen nuclei of glu 3 , where cu(ii ) 
 is coordinated to the carbonyl oxygen of ala 2 .
this suggests just a single histidine residue is coordinated 
 to cu(ii ) in component ii coordination .
comparison of eseem spectra 
 of cu(ii)a(116 ) 
 complex at ph 8.7 and the two - imidazole complex .
the inset shows the 
 increase of component ii cu(ii ) contribution in the presence of zn(ii ) .
the normalized integrated intensity 
 ( n - eseem / h - eseem ) of a(116 ) 
 is 17 compared to 21 and 
 22 for the two - imidazole complex and the phgggw , respectively .
the 
 lower value for a(116 ) is possibly due to the smaller 
 number of protons that interact with the cu(ii ) center .
the cu(ii ) 
 centers in the model complexes are solvent accessible . in a(116 ) 
 the solvent 
cu(ii ) interaction may be restricted because of 
 the neighboring amino acids . in our analysis
we have normalized the 
 integrated area of the h - eseem peak to be the same for 
 all spectra .
therefore , we may be underestimating the n - eseem integrated intensity for a(116 ) .
nevertheless , 
 the normalization method does suggest that cu(ii ) is coupled to two n - eseem active nuclei , not one or three .
hence , we can answer 
 the crucial question of the number of histidines coordinated to component 
 ii .
three - pulse eseem spectroscopy was used in conjunction 
 with isotopic substitution to determine the coordination of component 
 ii .
specifically the aim of these experiments was to provide more 
 insight into the proportions of histidine residues involved in component 
 ii coordination .
two histidine residues at a time are isotopically labeled 
 with n. upon n substitution , the modulation 
 depths of the frequencies due to eseem active n nuclei 
 will decrease .
this decrease is because the single quantum transition 
 of n nuclei does not substantially contribute to the 
 eseem signal .
we compare the integrated intensities of nonlabeled and n - labeled variants . as the modulation depth of h frequency 

is not affected by the n substitution , the h eseem peak is used to normalize the integrated intensity of n eseem . because two of the three histidine residues are 
 labeled , the n eseem signal intensity results only from 
 the single nonlabeled histidine .
this provides a direct method to 
 determine the extent to which each histidine residue is involved in 
 component ii .
all the samples were at ph 8.7 , and the experiments 
 were carried out at 3355 g , which corresponded to the maximum signal 
 in the echo detected field sweep .
the eseem intensities are integrated 
 between 0 and 11 mhz in all the eseem spectra collected ( figure 7 ) .
the integrated eseem intensity of the his 6,13-labeled 
 variant was 40% of that of the nonlabeled variant .
in the 
 his 6,13 variant , his 14 is the only labeled histidine residue and 
 the n eseem intensity results from only his 14 .
likewise , 
 his 13 contributes 40% and his 6 , 20% , toward the component 
 ii cu(ii ) coordination ( table 2 ) .
the 
 integrated intensities are tabulated in the supporting 
 infomation ( table s2 ) . to further confirm the proportions of 
 the histidine residues
, we performed the experiments using single - labeled 
 variants in which only one histidine is labeled at a time .
the decrease 
 in the signal intensity in the n eseem region with respect 
 to the nonlabeled variant indicates the extent of the involvement 
 of the labeled histidine in the coordination .
the eseem spectra for 
 single - labeled variants are shown in figure s4 , supporting infomation .
the integrated n eseem 
 intensities ( table s3 , supporting infomation ) for single - label variants show the similar pattern of his 14  
 his 13 > his 6 as observed with the double - label variants .
three - pulse 
 eseem spectra of the nonlabeled and single n - labeled 
 a(116 ) variants mixed with equimolar amounts 
 of cu(ii ) at ph 8.7 .
the decrease in intensity below 8 mhz in n - labeled a(116 ) variants gives the contribution 
 of each histidine residue for component i in a(116)cu(ii ) . 

the inset shows an expanded view of the 06 mhz region for 
 the labeled peptides .
this trend 
 can be rationalized by the pka values 
 of the histidine side chains .
his 6 has a pka of 7.1 , while for his 13 and 14 the pka values are 7.7 and 7.8 , respectively .
as the imidazole ring in his 6 has a lower pka value , ring nitrogens will be deprotonated at lower 
 ph values than in his 13 and 14 .
when the ph is raised , 
 his 13 and 14 rings become more accessible for cu(ii ) coordination . 

hence the proportions of his 13 and 14 are increased with the increase 
 in ph .
these results are in accordance with an x - ray examination of 
 a(116 ) , which suggests that his 13 and his 14 are readily 
 accessible for metal ion coordination .
the design of a curcumin scaffold was discussed in this work , which 
 is used to compete for cu(ii ) coordination with the his 13his 
 14 dyad .
hence , 
 we suggest only one histidine residue is involved in component 
 ii , with a preference to his 13 and his 14 over his 6 .
the other residues 
 involved in the component ii coordination are the carbonyl oxygen 
 of ala 2 , the n - terminus ( asp 1 ) , and the amide nitrogen of ala 2 .
the peak around 2.8 mhz in the eseem spectra 
 is indicative of backbone coordination and further confirms the involvement 
 of the peptide backbone in component ii coordination .
his 13 
 and his 
 14 equatorially coordinate to cu(ii ) more than does his 6 .
our eseem results performed using both the double 
 and single n - labeled histidine residues indicate that 
 his 13 and his 
 14 have a higher preference for the equatorial coordination position 
 in cu(ii ) component ii coordination . chemically , component ii cu(ii ) 
 coordination is really interesting as zn(ii )
biologically , it is important 
 to understand the component ii coordination , as component ii may be 
 the most significant cu(ii ) coordination in vivo , as zn(ii ) coexists 
 with cu(ii ) .
the insight into the component ii coordination , more 
 importantly the proportions of subcomponents , will shed light on understanding 
 the role of metal ions in alzheimer s disease .
we experimentally validated the use of eseem intensities to quantify 
 the number of n nuclei coupled to a cu(ii ) ion . a monotonic 
 increase in the n eseem intensities
were observed for 
 the model complexes synthesized with different numbers of imidazole 
 rings .
then , the validity of the method was tested with two well - characterized 
 cu(ii ) binding peptides .
we used our method to solve an important 
 structural problem in acu(ii ) complex .
we determined 
 that only a single histidine residue is coordinated to cu(ii ) ion 
 in component ii in a. finally , in component ii , cu(ii ) uses 
 his 13 and his 14 as an equatorial ligand over his 6 .
the proportions 
 of the three histidine residues in cu(ii ) coordination can be rationalized 
 by the pka values of the histidine side 
 chains .
shedding light into the component ii coordination was critical 
 as component ii might be the dominant cu(ii ) coordination mode of 
 a in vivo .
 we 
 validate the use of eseem to predict the number of 14n 
 nuclei coupled to a cu(ii ) ion by the use of model complexes and 
 two small peptides with well - known cu(ii ) coordination . 
 we apply this 
 method to gain new insight into less explored aspects of cu(ii ) coordination 
 in amyloid- ( a ) . 
 a has two coordination modes 
 of cu(ii ) at physiological ph . 
 a controversy has existed regarding 
 the number of histidine residues coordinated to the cu(ii ) ion in 
 component ii , which is dominant at high ph ( 8.7 ) values . 
 importantly , 
 with an excess amount of zn(ii ) ions , as is the case in brain tissues 
 affected by alzheimer s disease , component ii becomes the dominant 
 coordination mode , as zn(ii ) selectively substitutes component i bound 
 to cu(ii ) . 
 we confirm that component ii only contains single histidine 
 coordination , using eseem and set of model complexes . 
 the eseem experiments 
 carried out on systematically 15n - labeled peptides reveal 
 that , in component ii , his 13 and his 14 are more favored as equatorial 
 ligands compared to his 6 . revealing molecular level details of subcomponents 
 in metal ion coordination is critical in understanding the role of 
 metal ions in alzheimer s disease etiology . 