Patent Application: US-68423510-A

Abstract:
mutants of human fgf - 1 are disclosed having increased stability and mitogenic potency . in the fgf - 1 polypeptide , primarily residue 12 is substituted with cysteine and / or residue 134 is substituted cysteine , valine or threonine to render the polypeptide more stable and / or to increase its mitogenecity .

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 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 . fig1 through 6 illustrate various aspects of the present disclosure regarding human fibroblast growth factor - 1 ( fgf - 1 ), which is a potent human mitogen for a variety of cell types , including vascular endothelial cells , and can stimulate such cells to develop neovasculature capable of relieving ischemia . for this reason , fgf - 1 is an angiogenic factor with potential applicability in “ angiogenic therapy ”. 1 - 3 fgf - 1 belongs to the β - trefoil superfold . 4 , 5 this molecular architecture is characterized by a pseudo - 3 - fold axis of structural symmetry , with the repeating motif being a pair of anti - parallel β - strands , known as the “ β - trefoil fold ”. these repeating structural motifs comprise a total of 12 β - strands that associate to form a six - stranded β - barrel capped at one end by three β - hairpins ( forming the “ β - trefoil ” superfold ; fig1 ). residue positions 13 - 17 ( using the 140 amino acid form of fgf - 1 numbering scheme ) of the n - termini ( β - strand 1 ), and 131 - 135 of the c - termini ( β - strand 12 ), hydrogen bond with each other as a pair of anti - parallel β - strands within the six - stranded β - barrel , closely juxtaposing the two termini . when considering the three - fold symmetry of the overall architecture , the n - and c - termini are structurally related to two β - hairpin turns at positions 49 - 52 and 90 - 93 ( fig1 ). thus , the termini in the native structure represent a break in the mainchain continuity that forms the β - barrel . an analysis of correlated anisotropic thermal factors in a 1 . 10 å atom - resolution x - ray structure of fgf - 1 , has identified the n - and c - termini β - strands ( β - strands 1 and 12 , respectively ) as demarcating a boundary of domain motion within fgf - 1 6 . in the solution nmr structure of fgf - 1 the interaction between β - strands 1 and 12 is only consistently defined through residue position 133 in β - strand 12 , and the remaining positions 134 - 135 appear largely disordered 7 . thus , these data are consistent with the n - and c - termini β - strand interaction representing a region of structural weakness in fgf - 1 and therefore potentially contributing to the unfolding process . of additional interest , quenched - flow hydrogen exchange experiments with fgf - 1 have shown that the hydrogen bonds linking the n - and c - termini anti - parallel β strands appear to be the first detectable event in the folding of fgf - 1 , and may provide a structural framework for subsequent folding events . 8 thus , in addition to unfolding , the interaction of the n - and c - termini β - strands may be a key contributor to the folding of fgf - 1 . in an effort to study the contribution of the n - and c - termini β - strands to the stability and folding of fgf - 1 , cys mutations were introduced into each β - strand with the intention of linking them through a disulfide bond . in this case , stability and folding studies under oxidizing and reducing conditions might elucidate the contribution of the n - and c - termini β - sheet formation to these processes . two potential sites for such pair - wise mutations were identified at positions 12 and 134 , and 13 and 135 , respectively . these two pair wise cys mutants were constructed and initial stability studies were performed under oxidizing conditions . the cys13 / cys135 mutant exhibited a substantial decrease in stability and was not studied further . in contrast , the cys12 / cys134 mutant exhibited a substantial increase in stability , suggesting that the introduced disulfide bond had stabilized the structure . however , repeating the stability studies under reducing conditions resulted in a further gain in stability . therefore , the increase in stability for the cys12 / cys134 mutant was due to the substitution of lys12 and / or pro134 by cys and not to disulfide bond formation . as a consequence of this initial result , additional thr and val point mutations were constructed at positions 12 and 134 to probe the nature of the stability increase afforded by the cys mutations . the results of these studies show that the cys residue , in each case , is not unique and similar or greater increases in stability can be realized with either thr or val mutations . isothermal equilibrium denaturation , folding and unfolding kinetics , and x - ray structural studies have been utilized in characterizing the effects of cys , thr and val mutations at positions 12 and 134 in fgf - 1 . the results show that mutations at both positions 12 and 134 contribute to increased stability , with position 12 mutations primarily increasing the rate of folding , and position 134 mutations primarily decreasing the rate of unfolding . the combined position 12 and 134 val mutation also exhibits a 30 - fold increase in mitogenic potency and may find useful application as a “ second generation ” form of fgf - 1 in angiogenic therapy . val mutations at the symmetry - related positions of residues 12 and 134 were also studied and in one case ( position 95 ) provide a substantial additional increase in stability . a combined mutation , involving val mutations at five positions , and introducing a three - fold symmetric constraint at two positions within the fgf - 1 structure , results in an increase in stability that doubles the original value of the δg of unfolding . this combined mutation is , however , functionally inactive . the results provide additional support to our hypothesis 9 that a symmetric primary structure within a symmetric superfold is a solution to , and not a constraint upon , the protein folding problem . furthermore , the results also support the “ function / stability trade - off ” hypothesis 10 - 14 , and lead us to propose that one property of the β - trefoil superfold ( and presumably all the protein superfolds ) is the capacity for profound thermal stability , permitting a wide range of adaptive radiation in function . 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 fgf - 1 was chosen as the reference protein for the current set of mutations , and is referred to as wt * in this report . 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 guanidine hydrochloride ( 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 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 * 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 ) 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 decay at all denaturant concentrations . the folding kinetic data also exhibit an excellent fit to a single exponential model , but only for denaturant concentrations above approximately 0 . 6m guhcl . below this concentration , the folding kinetic data exhibit 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 ( mf [ d ])+ ln ( k f0 exp ( m u [ d ])) ( 4 ) where k f0 and k u0 are the folding and unfolding rate constants , respectively , extrapolated to 0m denaturant , mf 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 , 100 mm nacl , 10 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 pro134 cys mutant which grew from 3 . 6m sodium formate with no added ammonium sulfate . diffraction data for all mutants except pro134 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 .). pro134 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 : 1jqz ) 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 r free 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 % co2 supplementation . at 80 % cell confluence , the cells were washed with 5 ml cold 0 . 14 m nacl , 5 . 1 mm kcl , 0 . 7 mm na2hpo4 , 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 × 104 cells / cm2 ( 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 hemocytometer ( 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 ( ec 50 value ), and is used for quantitative comparison of mitogenicity . the thermodynamic parameters for the fgf - 1 mutants are listed in table i . 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 lys12 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 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 . 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 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 *. diffraction - quality crystals were obtained for the lys12 cys , lys12 val , lys12 thr , lys12 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 ( c222 1 ) with two molecules in asymmetric unit . the pro 134 cys mutant crystallized in the monoclinic p2 1 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 2f o - f c 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 * cys117 val reference protein 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 . differential scanning calorimetry studies of k12v / c117v fgf - 1 and p134v / c117v fgf - 1 were conducted and compared to c117v fgf - 1 (“ wild - type ” fgf - 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 graph depicted in fig7 shows the derived free energy profile ( dg ) as a function of temperature for the above mutants and “ wild type ” fgf - 1 . 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 å 3 . 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 sã . 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 β - strand 4 , and not β - 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 χ2 angle . this alternative side chain orientation positions one of the leu δ 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 waals 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 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 β - 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 increases in mitogenicity ( table iv ). the lys 12 side chain does not directly interact with fgfr ( pdb accession 1 e0o ), 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 . the results shown in fig6 indicate 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 ( 1 )); 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 ). 35 the cys , val and thr mutations at position 12 exhibit closely - related effects as regards their substantial increase in stability . similarly , the cys , val and thr mutations at position 134 also exhibit closely - related substantial increases in stability . these similar mutagenic effects observed for the set of cys , val and thr amino acids reflect their related stereochemical properties . of the 20 common amino acids , the set of cys , val , and thr amino acids comprise a unique set : i . e . they are the only amino acids that contain at least a side chain gamma constituent , but no constituent beyond the gamma position ( i . e . no delta , epsilon , etc . constituent ). the x - ray structure analyses of the role of the side chain gamma constituent in increasing the protein stability is consistent with this interpretation . given these data , only representative single and double mutations involving positions 12 and 134 were deemed necessary to evaluate in the functional ( i . e . 3t3 mitogenic ) assay . all possible combinations of cys , val , and thr single and double mutations at positions 12 and 134 comprise a total set of 15 mutations ; however , based on the stability and structural data , we conclude that the single and double val mutants characterized in the mitogenic assay are appropriately representative of the different combinations of cys , val and thr mutations at these positions . the structure and stability data presented in this application allow us to predict the utility of the various cys , val and thr mutations at positions 12 and 134 . 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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