Patent Abstract:
the β - trefoil protein human fibroblast growth factor - 1 is made up of a six - stranded anti - parallel β - barrel closed off on one end by three β - hairpins , thus exhibiting a three - fold axis of structural symmetry . the n - and c - termini β - strands hydrogen bond to each other and are postulated from both nmr and x - ray structure data to represent a structurally - weakened region of the β - barrel . val mutations within the n - and c - termini β - strands are shown to stabilize the structure and to increase van der waals contacts by filling local cavities present within this region . mutations that increase van der waals contacts between both the n - and c - termini β - strands are generally associated with significant reductions in the unfolding kinetics , and also increase the cooperativity of unfolding . surprisingly , several mutant polypeptides herein disclosed greatly exceed the wild - type polypeptide in ability to stimulate human fibroblasts to proliferate .

Detailed Description:
the present invention will now be described more fully hereinafter with reference to the accompanying drawings , in which preferred embodiments of the invention are shown . unless otherwise defined , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains . although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention , suitable methods and materials are described below . any publications , patent applications , patents , or other references mentioned herein are incorporated by reference in their entirety . in case of conflict , the present specification , including any definitions , will control . in addition , the materials , methods and examples given are illustrative in nature only and not intended to be limiting . accordingly , this invention may , however , be embodied in many different forms and should not be construed as limited to the illustrated embodiments set forth herein . rather , these illustrated embodiments are provided so that this disclosure will be thorough and complete , and will fully convey the scope of the invention to those skilled in the art . other features and advantages of the invention will be apparent from the following detailed description , and from the claims . mutant construction and expression followed previously described procedures 15 - 17 . briefly , all studies utilized a synthetic gene for the 140 amino acid form of human fgf - 1 18 - 21 with the addition of an amino - terminal six residue “ his - tag ” to facilitate purification . 17 in the present study a cys117 → val mutant form of wild - type fgf - 1 was chosen as the reference protein for the current set of mutations , and is referred to as wt * in this report and shown herein as seq id no : 1 . the cys117 → val mutation has minimal effects upon stability , folding or function of fgf - 1 17 but eliminates a surface exposed cysteine residue that can form an intermolecular disulfide bond . the quikchange ™ site - directed mutagenesis protocol ( stratagene , la jolla , calif .) was used to introduce individual or combination mutations using mutagenic oligonucleotides of 25 to 31 bases in length ( biomolecular analysis synthesis and sequencing laboratory , florida state university ). all fgf - 1 mutants were expressed using the pet21a (+) plasmid / bl21 ( de3 ) escherichia coli host expression system ( invitrogen corp ., carlsbad calif .). mutant proteins were purified as previously described 17 using nickel - nitrilotriacetic acid ( ni - nta ) chromatography followed by affinity purification using heparin sepharose chromatography ( g . e . healthcare , piscataway n . j .). sites for cys mutations leading to potential disulfide bond formation were identified using the disulfide by design program 22 and the x - ray coordinates of wild - type fgf - 1 . 18 isothermal equilibrium denaturation by guhcl was quantified using fluorescence as the spectroscopic probe . fgf - 1 contains a single buried tryptophan residue ( trp107 ) that exhibits greater fluorescence quenching in the native versus denatured state . 15 , 18 the differential fluorescence between the native and denatured state has been used to quantify the unfolding of fgf - 1 , in excellent agreement with unfolding as monitored by cd spectroscopy . 15 , 23 fluorescence data were collected on a varian eclipse fluorescence spectrophotometer equipped with a peltier controlled temperature regulator at 298k and using a 1 cm path - length cuvette . protein samples ( 5 μm ) were equilibrated overnight in 20 mm ada , 100 mm nacl , 2 mm dtt ph 6 . 6 (“ ada buffer ”) at 298k in 0 . 1m increments of guanidine hcl ( guhcl ). triplicate scans were collected and averaged , and buffer traces were collected and subsequently subtracted from the protein scans . all scans were integrated to quantify the total fluorescence as a function of denaturant concentration . the general purpose non - linear least squares fitting program datafit ( oakdale engineering , oakdale , pa .) was used to fit the change in fluorescence versus guhcl concentration data to a six parameter two - state model 24 : f = f 0 ⁢ n + s n ⁡ [ d ] + ( f 0 ⁢ ⁢ d + ( s d ⁡ [ d ] ) ) ⁢ e - ( δ ⁢ ⁢ g ⁢ ⁢ 0 + m ⁡ [ d ] ) / rt 1 + e - ( δ ⁢ ⁢ g ⁢ ⁢ 0 + m ⁡ [ d ] ) / rt ( 1 ) where [ d ] is the denaturant concentration , f 0n and f 0d are the 0m denaturant molar ellipticity intercepts for the native and denatured state baselines , respectively , and s n and s d are the slopes of the native and denatured state baselines , respectively . δg 0 and m describe the linear function of the unfolding free energy versus denaturant concentration . the effect of a given mutation upon the stability of the protein ( δδg ) was calculated by taking the difference between the c m values for wt * ( seq id no : 1 ) and mutant proteins and multiplying by the average of the m values , as described by pace and scholtz 25 : δδ g =( c m wt * − c m mutant )( m wt * + m mutant )/ 2 ( 2 ) fig7 shows the results of differential scanning calorimetry studies of k12v / c117v fgf - 1 ( seq id no : 3 ) and p134v / c117v fgf - 1 ( seq id no : 9 ) in comparison to c117v fgf - 1 (“ wild - type ” fgf - 1 ; seq id no : 1 ). we performed thermal denaturation studies of the above mutants using differential scanning calorimetry ( dsc ). this method permits direct determination of the melting temperature ( melting transition midpoint ) of a protein . the k12v and p134v point mutations were made in a modified version of wild - type fgf - 1 that contains a cys to val mutation at position 117 . this mutation has no effect upon stability of the protein , however , it eliminates the possibility of disulfide - linked dimers of fgf - 1 ( which is problematic for dsc analysis ). the image in fig7 shows the derived free energy profile ( δg ) as a function of temperature for the above mutants and “ wild type ” fgf - 1 . this result shows that the k12v mutation increases the melting temperature by 16 . 9 ° c . and the p13v mutation increases the melting temperature by 15 . 7 ° c . this is similar to the increase in stability afforded by the addition of heparin ( see copeland ( 38 )); thus , these mutations may obviate the need to add heparin in the formulation of fgf - 1 ( saving considerable cost and avoiding concerns of infectious agents ( since heparin is derived from pig tissue )). initial studies using manual mixing indicated that the relaxation times for folding were more appropriate for stopped - flow data collection . denatured protein samples were prepared by overnight dialysis against ada buffer containing either 2 . 5 m or 3 . 0 m guhcl ( depending upon the overall stability of the mutant ). all folding kinetic data were collected using a kintek sf2000 stopped - flow system ( kintek corp ., austin tex .). folding was initiated by a 1 : 10 dilution of 40 μm denatured protein into ada buffer with denaturant concentrations varying in increments of 0 . 05 m or 0 . 1 m , to the midpoint of denaturation as determined by the above described isothermal equilibrium denaturation measurements . the data collection strategy was designed to span approximately five half - lives , or & gt ; 97 % of the expected fluorescence signal change between the fully denatured and native states . unfolding kinetics measurements were performed using a manual mixing technique . protein samples (˜ 30 μm ) were dialyzed against ada buffer overnight at 298k . unfolding was initiated by a 1 : 10 dilution into ada buffer with a final guhcl concentration of 1 . 5 to 5 . 5m in 0 . 5m increments . all unfolding data were collected using a varian eclipse fluorescence spectrophotometer equipped with a peltier controlled temperature unit at 298k . data collection times for each protein were designed so as to quantify the fluorescence signal over 3 - 4 half - lives , or & gt ; 93 % of the total expected amplitude . the folding and unfolding characteristics of fgf - 1 have previously been described in detail . 26 briefly , the unfolding kinetic data exhibits an excellent fit to single exponential decayat all denaturant concentrations . the folding kinetic data also exhibits an excellent fit to a single exponential model , but only for denaturant concentrations above approximately 0 . 6 m guhcl . below this concentration , the folding kinetic data exhibits bi - exponential properties ; with the slow phase being generally independent of denaturant concentration . the fast phase of this biexponential folding regime lies on the extrapolated region of the single - exponential folding data . thus , the folding constant is derived from a fit to the mono - exponential region and the fast phase of the bi - exponential region . the δg values derived from the folding and unfolding kinetic data are in excellent agreement with the values obtained from isothermal equilibrium denaturation data , as well as differential scanning calorimetry 26 . both folding and unfolding kinetic data were collected in triplicate at each guhcl concentration ; data from at least three separate experiments were averaged in each case . the kinetic rates and amplitudes versus denaturant concentration were calculated from the time dependent change in tryptophan fluorescence using a single exponential model : where l ( t ) is the intensity of fluorescent signal at time t , a is the corresponding amplitude , k is the observed rate constant for the reaction and c is the asymptote of the fluorescence signal . folding and unfolding rate constant data were fit to a global function describing the contribution of both rate constants to the observed kinetics as a function of denaturant (“ chevron ” plot ) as described by fersht 27 : ln ( k obs )= ln ( k f0 exp ( m f [ d ])+ k u0 exp ( m u [ d ])) ( 4 ) where k f0 and k u0 are the folding and unfolding rate constants , respectively , extrapolated to 0m denaturant , m f and m u are the slopes of the linear functions relating ln ( k f ) and ln ( k u ), respectively , to denaturant concentration , and [ d ] is the denaturant concentration . crystallization of fgf - 1 mutants , x - ray data collection , refinement and cavity calculations purified protein for crystallization trials was dialyzed against 50 mm sodium phosphate , 1 00 mm nacl , 1 0 mm ammonium sulfate , 2 mm dtt ph 7 . 5 (“ crystallization buffer ”) and concentrated to 10 - 16 mg / ml . crystals were grown at room temperature using the hanging - drop vapor diffusion method with 7 μl drop size and 1 ml of reservoir solution . diffraction quality crystals grew from reservoirs containing 3 . 2 - 4 . 3 m sodium formate and 0 . 25 - 0 . 5 m ammonium sulfate , with the exception of the pro 134 → cys mutant which grew from 3 . 6 m sodium formate with no added ammonium sulfate . diffraction data for all mutants except pro 134 → cys , was collected at the southeast regional collaborative access team ( ser - cat ) 22 - bm beam line ( λ = 1 . 00 å ) at the advanced photon source , argonne national laboratory , using a marccd225 detector ( mar usa , evanston , ill .). pro 134 → cys mutant diffraction data was collected using an in - house rigaku ru - h2r copper rotating anode ( λ = 1 . 54 å ) x - ray generator ( rigaku msc , the woodlands , tex .) coupled to an osmic purple confocal mirror system ( osmic , auburn hills , mich .) and a marccd165 detector ( mar usa , evanston , ill .). in all cases , crystals were mounted and maintained in a stream of gaseous nitrogen at 100 k . diffraction data were indexed , integrated and scaled using the hkl2000 software . 28 , 29 his - tagged wild - type fgf - 1 ( pdb code : 1 jqz ) was used as the search model in molecular replacement using the cns software suite . 30 model building and visualization utilized the o molecular graphics program . 31 structure refinement utilized the cns software suite , with 5 % of the data in the reflection files set aside for rfree calculations 32 . quantification of solvent - excluded cavities with the refined mutant structures was performed using the msp software package . 33 the mitogenic activity of certain mutants was evaluated by a cultured fibroblast proliferation assay . nih 3t3 fibroblasts were initially plated in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) ( american type culture collection , manassas va .) supplemented with 10 % ( v / v ) newborn calf serum ( ncs ) ( sigma , st louis mo . ), 100 units of penicillin , 100 mg of streptomycin , 0 . 25 mg of fungizone ™ and 0 . 01 mg / ml of gentamicin ( gibco , carlsbad calif .) (“ serum - rich ” medium ) in t75 tissue culture flasks ( fisher , pittsburgh pa .). the cultures were incubated at 37 ° c . with 5 % co 2 supplementation . at 80 % cell confluence , the cells were washed with 5 ml cold 0 . 14 m nacl , 5 . 1 mm kcl , 0 . 7 mm na 2 hpo 4 , 24 . 8 mm trizma base , ph 7 . 4 ( tbs ) and subsequently treated with 5 ml of a 0 . 025 % trypsin solution ( invitrogen corp ., carlsbad calif .). the trypsinized cells were subsequently seeded in t25 tissue culture flasks at a density of 3 . 0 × 10 4 cells / cm 2 ( representing 20 % confluence ). cell synchronization was initiated by serum starvation in dmem with 0 . 5 % ncs , 100 units of penicillin , 100 mg of streptomycin , 0 . 25 mg of fungizone ™ and 0 . 01 mg / ml of gentamicin (“ starvation ” medium ). cultures were incubated for 48 hours at 37 ° c ., the medium was then decanted and replaced with fresh medium supplemented with fgf - 1 ( 0 - 10 μg / ml ), and the cultures incubated for an additional 48 hours . after this incubation , the medium was decanted and the cells were washed with 1 ml of cold tbs . 1 ml of 0 . 025 % trypsin was then added to release the cells from the flask surface , and 2 ml of serum - rich medium was added to dilute and inhibit the trypsin . the cells were counted using a hemacytometer ( hausser scientific partnership , horsham pa .). experiments were performed in quadruplicate and the cell densities were averaged . the relationship between the cell number and log concentration of added growth factor was fit to a sigmoid function . the midpoint of the fitted sigmoid function represents the concentration of added growth factor necessary to achieve 50 % stimulation ( ec50 value ), and is used for quantitative comparison of mitogenicity . all mutants , except those containing glu87 → val , purified with high yield (˜ 65 mg / l ). proteins containing the glu87 ˜ val mutation appeared to have a lower solubility , resulting in some precipitation during purification and with associated lower yield (˜ 35 - 40 mg / l ). the thermodynamic parameters for the fgf - 1 mutants are listed in table i . each of the polypeptide sequences disclosed in table i is also identified according to seq id no . the polypeptide sequences found to be most active in stimulating proliferation of fibroblasts are seq id nos : 2 - 4 . the standard error of δg from multiple analyses is approximately 1 . 0 kj / mol ( 0 . 24 kcal / mol ), which is also the typical magnitude of the standard deviation of the fit to the 2 - state model ( data not shown ). thus , mutational effects upon stability can be reliably measured for values greater than 1 kj / mol , consistent with previous reports , and the mutational effects upon stability are larger than this standard error in each case . the substitution of lys 12 by cys , thr or val provides a substantial increase in stability of between − 6 . 9 to − 8 . 1 kj / mol . the highest midpoint of denaturation is observed for the val mutant ( 1 . 53 m ); however , a slight reduction in the δg versus denaturant m - value for the val mutant in comparison to cys results in a somewhat higher δg value for cys when extrapolated to 0m denaturant ( table i ). overall , therefore , the cys and val table i thermodynamic parameters for fgf - 1 mutants derived from isothermal equilibrium denaturation studies in ada buffer ( see text for details ). δg a m - value cm δδg b mutant ( kj / mol ) ( kj / mol m ) ( m ) ( kj / mol ) wt * 21 . 5 ± 0 . 8 19 . 5 ± 0 . 7 1 . 10 ± 0 . 02 lys 12 → cys / 36 . 2 ± 0 . 5 19 . 6 ± 0 . 3 1 . 84 ± 0 . 01 − 14 . 5 pro1 34 → cys lys 12 → cys / 30 . 5 ± 0 . 7 18 . 3 ± 0 . 4 1 . 66 ± 0 . 01 − 10 . 6 pro134 → cys ( oxidized ) lys 12 → cys 29 . 2 ± 0 . 7 19 . 4 ± 0 . 6 1 . 50 ± 0 . 01 − 7 . 8 lys 12 → thr 26 . 2 ± 0 . 9 17 . 8 ± 0 . 6 1 . 47 ± 0 . 02 − 6 . 9 lys12 → val 27 . 7 ± 0 . 5 18 . 1 ± 0 . 3 1 . 53 ± 0 . 01 − 8 . 1 pro134 → cys 26 . 9 ± 0 . 7 19 . 1 ± 0 . 5 1 . 41 ± 0 . 01 − 6 . 0 pro134 → thr 26 . 9 ± 0 . 4 19 . 9 ± 0 . 3 1 . 34 ± 0 . 01 − 4 . 7 pro134 → val 28 . 8 ± 0 . 7 19 . 3 ± 0 . 5 1 . 49 ± 0 . 01 − 7 . 6 lys 12 → val / 37 . 6 ± 0 . 6 18 . 5 ± 0 . 3 2 . 03 ± 0 . 01 − 17 . 7 pro1 34 → val lys12 → val / 34 . 1 ± 1 . 0 18 . 3 ± 0 . 6 1 . 86 ± 0 . 02 − 14 . 4 asn95 → val leu46 → val / 27 . 7 ± 0 . 7 19 . 4 ± 0 . 5 1 . 43 ± 0 . 02 − 6 . 4 pro 134 → val glu87 → val / 27 . 7 ± 0 . 9 18 . 8 ± 0 . 6 1 . 47 ± 0 . 02 − 7 . 0 pro 134 → val leu46 → val / 28 . 8 ± 1 . 3 18 . 7 ± 0 . 8 1 . 54 ± 0 . 02 − 8 . 4 glu87 → val / pro134 → val lys 12 → val / 40 . 6 ± 1 . 3 16 . 6 ± 0 . 6 2 . 44 ± 0 . 02 − 24 . 1 leu46 → val / glu87 → val / asn95 → val / pro134 → val * reference protein for all studies is fgf - 1 cys117 → val mutant ( see text ). a äg value extrapolated to 0 m denaturant . b δδg = ( c mwt * − c m mutant )( m wt * + m mutant )/ 2 as described by pace and scholtz 25 ; a negative value of { umlaut over ( aa )} g indicates a more stable mutation . error is the standard deviation of multiple data sets . mutants appear to be approximately equivalent in stability , with thr slightly less so ( but still stabilizing the protein by approximately − 7 . 0 kj / mol ). the substitution of pro 134 by cys , thr or val also provides a significant increase in stability of between − 4 . 7 to − 7 . 6 kj / mol . the highest midpoint of denaturation is observed for the val mutant ( 1 . 49 m ). in the case of position 134 mutations , the δg versus denaturant m - value is not substantially altered ( table i ), and extrapolation of δg to 0m denaturant similarly identifies the val mutant as the most stable at this position . combining val mutations at positions 12 and 134 results in a − 17 . 7 kj / mol increase in stability . the simple sum of the individual point mutations predicts an increase in stability of − 15 . 7 kj / mol ; thus , the effects of the combined mutation appear to be largely additive in nature , with the possibility of cooperative interactions providing a modest − 2 . 0 kj / mol of additional stability . a val mutation at position 95 was constructed in the lys 12 - val mutant background . position 95 is related to position 12 by the three - fold pseudo - symmetry inherent in the fgf - 1 architecture ( i . e . the β - trefoil superfold ). in reference to the lys12 → val mutant , the val mutation at position 95 stabilizes the protein by − 6 . 3 kj / mol ( table i ), a magnitude similar to that of the lys 12 → val mutation . individual val mutations at positions 46 and 87 were constructed in the pro 134 → val mutant . positions 46 and 87 are related to position 134 by the three - fold pseudo - symmetry in the fgf - 1 architecture . in reference to the pro 134 → val mutant , the val mutation at position 46 destabilizes the protein by a modest + 1 . 2 kj / mol , while the val mutation at position 87 is essentially neutral with regard to stability ( table i ). a combination of these val mutations at positions 46 and 87 was constructed in the pro 134 → val mutant . a simple sum of the individual mutational effects of the val 46 , 87 and 134 mutations predicts an overall increase in stability of − 5 . 8 kj / mol in comparison to the wt * protein ; however , the actual combination mutant exhibits an increase in stability of − 8 . 4 kj / mol . thus , the effects upon stability for these combined mutations are essentially additive , but with − 2 . 6 kj / mol of potential cooperativity . finally , the val mutations at positions 12 and 95 ( which constrains symmetry - related positions 12 , 54 and 95 to val ) and positions 46 , 87 and 134 ( which constrains these symmetry - related positions to val ) were combined into a single mutation . the summation of the stability effects of the position 12 and 95 mutations , and position 46 , 87 and 134 mutations , predicts an overall increase in stability of − 22 . 8 kj / mol ( table i ). the actual combination mutant exhibits an increase in stability of − 24 . 1 kj / mol ; therefore , the mutational effects of these two sets of symmetric val mutations appear to be essentially additive . the results of the folding and unfolding kinetic analyses are listed in table ii . the cys , thr , and val mutations at position 12 stabilize the protein by primarily increasing the folding rate constant ( 4 to 10 - fold ) with comparatively less - significant ( 2 - fold or less ) reduction in the unfolding rate constant . these alterations in the folding and unfolding rate constants are associated with minimal changes in either the folding or unfolding kinetics “ m values ”. the results of the cys , thr , and val mutations at position 134 upon the folding and unfolding rate constants are a bit more complex . the cys mutation achieves its increase in stability primarily through an 8 - fold increase in the folding rate constant , and less than 2 - fold decrease in the unfolding rate constant . thus , the stability increase for cys mutations at positions 12 and 134 are due to similar effects upon folding and unfolding kinetic rate constants ( i . e . primarily an increase in folding rate constant ). the thr mutation at position 134 achieves its increase in stability through an equivalent 2 - fold increase in folding rate constant and 2 - fold decrease in unfolding rate table ii folding and unfolding kinetic parameters for wt * and mutant fgf - 1 proteins . k f m f k u m u mutant ( s − 1 ) ( m − 1 ) ( 1 × 10 − 3 s − 1 ) ( m − 1 ) wt * 3 . 75 − 6 . 61 0 . 71 0 . 47 lys 12 → cys 15 . 0 − 6 . 38 0 . 27 0 . 69 lys 12 → thr 28 . 8 − 5 . 94 0 . 62 0 . 49 lys 12 → val 36 . 9 − 6 . 08 0 . 56 0 . 55 pro134 → cys 29 . 1 − 6 . 84 0 . 41 0 . 56 pro 134 → thr 7 . 15 − 6 . 17 0 . 43 0 . 52 pro134 → val 6 . 12 − 4 . 96 0 . 10 0 . 86 lys 12 → val / pro 134 → val 125 − 6 . 06 0 . 09 0 . 86 lys 12 → val / asn95 → val 161 − 6 . 88 0 . 15 1 . 06 leu46 → val / pro 134 → val 8 . 04 − 6 . 18 0 . 53 0 . 61 leu46 → val / glu87 → val / 25 . 4 − 6 . 49 0 . 70 0 . 57 pro 134 → val lys 12 → val / leu46 → val / 1 , 350 − 5 . 51 0 . 11 1 . 10 glu87 → val / asn95 → val / pro 134 → val * reference protein for all studies is fgf - 1 cys117 → val mutant ( see text ). constant . the val mutation at position 134 achieves its stability increase primarily through a 10 - fold decrease in unfolding rate constant , but also through an associated 6 - fold increase in folding rate constant . furthermore , the val mutation is associated with a 2 - fold increase in unfolding kinetics “ m value ” ( which is not observed in either the cys or thr mutation ; table ii ). the double val mutant at positions 12 and 134 exhibits the 10 - fold reduction in unfolding rate constant displayed by the val mutation at position 134 , as well as a 33 - fold increase in folding rate constant ( an enhancement of the 10 - fold increase in folding rate constant exhibited by the val mutant at position 12 ). this double mutant retains the 2 - fold increase in unfolding kinetics “ m value ” ( in comparison to wt *) displayed by the val mutation at position 134 ; there is no substantial change in the folding kinetics “ m value ” in comparison to wt *. the asn95 → val mutant achieves its increase in stability via a combination of a 4 - fold increase in folding rate constant and a 3 - fold decrease in unfolding rate constant ( comparing the lys 12 → val / asn95 → val double mutant to the lys12 → val as control ; table ii ). this mutation is also notable in that there is a 2 - fold increase in the unfolding kinetics “ m value ”. although the combined stability effects of the leu46 → val / glu87 → val double mutation result in a modest decrease in stability , the folding and unfolding rate constants exhibit a concomitant significant ( 4 to 7 - fold ) increase , indicating the folding transition state has been selectively stabilized . there are minimal effects upon either the folding or unfolding kinetics “ m value ” for these mutations . the final combination mutant , where the three - fold symmetry - related positions 12 , 54 and 95 , and 46 , 87 , and 134 are constrained to a val residue in each case , results in a 360 - fold increase in the folding rate constant and a 7 - fold decrease in unfolding rate constant . these changes in kinetic rate constants are associated with minimal effect upon folding “ m value ” but a 2 . 3 - fold increase in unfolding “ m value ” in comparison to the wt * protein . diffraction - quality crystals were obtained for the lys 12 → cys , lys 12 → val , lys12 → thr , lys 12 → val / asn95 → val and pro134 → cys mutants ( the majority of the position 134 mutations proving to be refractory to crystallization ). all structures were refined to acceptable crystallographic residuals and stereochemistry . crystallographic data collection and refinement statistics for the mutants are listed in table iii . all mutants , except pro 134 → cys , crystallized in the wt * orthorhombic space group ( c2221 ) with two molecules in asymmetric unit . the pro 134 cys mutant crystallized in the monoclinic p21 space group with four molecules in the asymmetric unit . these four molecules were successfully positioned using the molecular replacement method and wt * fgf - 1 as the search model . the 2fo − fc difference electron density was unambiguous at the mutation site ( s ), and the mutant structures could be accurately modeled in each case . the mitogenic activity ( ec 50 ) for representative fgf - 1 mutants is summarized in table iv ( the wt * cys 117 val reference protein , also shown as seq id no : 1 , is essentially identical to wild - type fgf - 1 in terms of stability , folding and mitogenic activity ). the cys and val mutations at position 12 are approximately equivalent to each other in terms of mitogenic activity , and both are approximately 15 times more potent than wt * fgf - 1 . in contrast , while the cys and val mutations at position 134 are similarly equivalent to each other in terms of mitogenic potential , they exhibit only a modest increase in mitogenic activity compared to wt * ( table iv ). the combination val mutation at positions 12 and 134 appears to be largely additive , exhibiting an approximately 30 - fold increase in mitogenic activity compared to wt *. the val mutation at position 95 exhibits a substantial ˜ 1000 - fold reduction in mitogenic activity . the val mutations at positions 46 and 87 are associated with relatively minor reductions in mitogenic activity , with position 46 slightly less active than wt *, and position 87 exhibiting a 2 - fold reduction in mitogenic activity . the combined val mutant at positions 12 , 46 , 87 , 95 and 134 exhibits no detectable mitogenic activity at concentrations up to 104 ng / ml , with higher protein concentrations observed to be toxic to 3t3 fibroblasts . the x - ray structure of wild - type fgf - 1 exhibits two small solvent - excluded cavities , detectable using a 1 . 2 å radius probe , in the region of residues 12 , 95 and 134 ( fig2 ) and these appear to be key to understanding the effects of the mutations at these positions . one cavity , adjacent to position 12 , and bounded by residues 14 , 44 and 46 , has a volume of 9 å3 ; the other cavity , adjacent to position 134 , and bounded by residue positions 14 , 95 and 97 , has a volume of 8 å3 . the wt * lys residue at position 12 adopts a χ1 angle of − 60 ° ( gauche +), which orients the lys12 side chain away from the adjacent cavity . however , the mutant cys residue at position 12 adopts a χ1 angle of + 60 ° ( gauche −) which positions the side chain sã towards the nearby cavity ( fig3 ). both the thr and val mutations at position 12 adopt rotamer angles that orient a side chain a methyl group in the same position as the cys sγ . thus , each of these small side chains is oriented so as to fill the adjacent cavity with a non - polar moiety . the lys12 does not appear capable of adopting a gauche − rotamer ( and filling the adjacent cavity ) due to resulting steric clashes with adjacent residue leu46 . in filling this adjacent cavity , the cys , thr or val residues are oriented to participate in van der waals contacts with residues in adjacent strand 4 , and not - strand 12 . thus , the observed increase in stability with the position 12 mutants does not appear to be associated with stabilizing interactions between the n - and c - termini β - strands . the x - ray structure of the cys mutation at position 134 provides an opportunity to understand the structural basis of the increase in stability for mutations at this position . the cys residue adopts a rotamer angle of − 60 ° ( gauche +) ( fig4 ). while generally oriented towards the cavity adjacent to position 134 , the mutant cys sγ does not appreciably reduce the size of the cavity . however , in response to the introduction of the cys at position 134 , the adjacent residue leu14 adopts an alternate + 2 angle . this alternative side chain orientation positions one of the leu a methyl groups towards the cavity adjacent to position 12 ; the result being that this cavity is no longer detectable using a 1 . 2 å radius probe . thus , the mutations at position 134 are capable of reducing the overall cavity space within the local region and increasing van der waals contacts between β - strand 1 and β - strand 12 ( i . e . the n - and c - termini ). in the x - ray structure of the combined val mutations at positions 12 and 95 , the val at position 12 behaves the same as the val 12 point mutation , and fills the adjacent cavity ( fig5 ). in response to the val mutation at position 95 , the pro side chain at position 134 shifts inward towards the cavity adjacent to this position , with the result that it is no longer detectable using a 1 . 2 å radius probe . this structural adjustment results in greater van der weals interactions between residue position 134 and adjacent residues , including leu14 on β - strand 1 . thus , the val mutation at position 95 also has the result of improving the van der waals interaction between β - strands 1 and 12 ( i . e . the n - and c - termini ). mutations at position 95 and 134 , but not position 12 , are unique in increasing the unfolding kinetics “ m value ” ( i . e . cooperativity of unfolding ; fig6 ) as well as decreasing the overall unfolding rate constant ( table ii ). an interpretation for an increase in the unfolding kinetics “ m value ” is that the mutation has introduced stabilizing interactions in the native structure , but not in the folding transition state , as would be expected if additional hydrophobic contacts had been formed in the native structure 34 . however , the position 12 mutations have similarly introduced additional hydrophobic contacts in the native state , but have not affected the unfolding kinetics “ m value ” nor significantly decreased the rate of unfolding . thus , the distinction is that the mutations that have stabilized interactions between β - strands 1 and 12 are responsible for the decreased unfolding rate constant and increased cooperativity of unfolding . therefore , it is concluded that early events in the unfolding of fgf - 1 likely involve melting of the interaction between β - strands 1 and 12 . this interpretation is consistent with the previously described domain motion boundary in fgf - 1 involving these β - strands 6 , and the solution nmr data indicating partial melting of the interface of β - strand 1 and 12 in fgf - 1 at 298k 7 . stabilizing adjacent n - and c - termini β - strand interactions may prove to be a generally - useful approach to engineering increased thermal stability in - barrel structures , and appears capable of providing a substantial increase to the stability of the protein . the val mutations at positions 12 and 134 are approximately equivalent in their favorable contribution to the stability of the protein (˜ 8 . 0 kj / mol ). fgf - 1 exhibits relatively low thermal stability 15 , 35 , and mutations that stabilize the structure can increase the effective mitogenic potency , presumably due to longer functional half - life 9 . both of the val mutations at positions 12 and 134 appear more functionally active than wt *, although the position 12 mutation has a much more dramatic increase in mitogenicity ( table iv ). the lys 12 side chain does not directly interact with fgfr ( pdb accession 1e0o ), neither does pro134 . thus , the basis for the difference in mitogenic activity between the 12 and 134 val mutants ( given their near - identical stability increase ) is not fully understood . nonetheless , the combined lys12 val / pro134 val mutant exhibits the greatest mitogenic activity , approximately 30 times more potent than wt *, and is − 17 . 7 kj / mol more stable than wt *. such mutant forms of fgf - 1 may find application as “ second generation ” forms of fgf - 1 in angiogenic therapy for the treatment of ischemia 3 , 36 . following our long - standing interest in the role of primary structure symmetry within a symmetric superfold , the effects of val mutations at symmetry - related positions to residues 12 and 134 were also examined . the symmetry mates of position lys 12 are val54 and asn95 . position 54 is already a val residue , and so mutation of positions 12 and 95 to val constrain these three symmetry - related positions to val . although the val95 mutation provides a substantial − 6 . 3 kj / mol increase in stability ( table i ), it has ˜ 1 , 000 - fold reduction in mitogenic activity ( table iv ). analysis of the fgf receptor ( fgfr )— fgf - 1 complex ( pdb accession 1e0o ) shows that asn95 in fgf - 1 makes an important hydrogen bonding interaction with arg 251 in the d2 and d3 linker of fgfr 37 ; therefore , the observed lack of mitogenicity appears due to disruption of this key contact . the glu87 mutant is neutral in its effect upon stability ( table i ), but its mitogenicity is decreased by a factor of 3 ( table iv ). analysis of the fgf receptor ( fgfr )— fgf - 1 complex shows that glu87 in fgf - 1 makes a hydrogen bonding interaction with arg 255 of fgfr . disruption of this interaction has diminished the mitogenicity , but not the extent observed for the asn95 val mutation ; thus , the asn95 ( fgf - 1 )/ arg251 ( fgfr ) interaction appears to be of considerably greater importance in formation of the fgf - 1 / fgfr complex than the glu87 ( fgf - 1 )/ arg255 ( fgfr ) interaction . the quintuple fgf - 1 mutant ( lys 12 val / leu46 val / glu87 val / asn95 val / pro134 val ) is the most stable in the series of mutants studied . the increase in stability ( δδg ) is − 24 . 1 kj / mol ( table i ); given that the overall stability of wt * fgf - 1 ( δg ) is 21 . 5 kj / mol ( table i ) the quintuple mutant has more than doubled this value . this combination mutant is also the most symmetric , with a three - fold symmetry constraint imposed upon the primary structure ( val ) at positions 12 , 54 , 95 and 46 , 87 , 134 . thus , these results support our proposal that a symmetric primary structure within a symmetric superfold is a solution to , and not a constraint upon , the protein folding problem . 9 previously reported fgf - 1 symmetric constraint mutations have resulted in increases of − 16 . 1 kj / mol 9 , again , almost doubling the magnitude of δg for the wt * protein . we conclude , therefore , that the β - trefoil architecture is intrinsically capable of substantial thermal stability and , furthermore , that such stability is achievable within a design principle of a symmetric primary structure . in prior studies of symmetric fgf - 1 mutants that were more stable , it was noted that heparin - binding functionality ( but not mitogenicity ) was lost .&# 39 ; thus , heparin functionality fell into the category of a demonstrable “ function / stability trade - off ” 10 - 14 in the fgf - 1 architecture . in the present case , the increase in stability afforded by the asn95 val is associated with apparent loss of receptor binding functionality . therefore , it appears that any hypothetical symmetric form of fgf - 1 with substantially increased thermal stability would also likely be devoid of fgf - specific functionality ( and possibly any identifiable function ). we propose that the hypothesized capacity for extreme thermostability of the β - trefoil architecture underscores the selection of the β - trefoil architecture as one of the fundamental protein superfolds . given the evidence for a function / stability trade - off , diverse functional radiation requires a protein architecture with the capacity to offset a wide range of destabilizing mutations . conversely , a protein architecture with limited capacity for thermal stability is unlikely to be capable of diverse functional adaptation . in addition to the mutant polypeptide sequences previously described , and based upon stability studies comparing val , thr , and cys mutants , we expect equivalent effects among these mutations at position12 , and similarly , equivalent effects among these mutations at position 134 . therefore , the following polypeptide sequences are offered as prophetic examples of mutant polypeptides expected to have similarly enhanced stability and / or mitogenicity : accordingly , in the drawings and specification there have been disclosed typical preferred embodiments of the invention and although specific terms may have been employed , the terms are used in a descriptive sense only and not for purposes of limitation . the invention has been described in considerable detail with specific reference to these illustrated embodiments . it will be apparent , however , that various modifications and changes can be made within the spirit and scope of the invention as described in the foregoing specification and as defined in the appended claims . 1 . folkman , j ., angiogenesis : initiation and control . annals of the new york academy of science 1982 , 401 , 212 - 227 . 2 . thomas , k . a . ; 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