RELATED APPLICATION 
     This application claims priority from co-pending provisional application Ser. No. 60/865,030, which was filed on Nov. 9, 2006, and which is incorporated herein by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the field of molecular medicine and, more particularly, to mutant molecules artificially created to have increased stability and greatly enhanced mitogenicity for human fibroblasts. 
     BACKGROUND OF THE INVENTION 
     Human fibroblast growth factor-1 (FGF-1) 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;  FIG. 1 ). Residue positions β-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 ( FIG. 1 ). Thus, the termini in the native structure represent a break in the mainchain continuity that forms the (3-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 βstrands 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 Cys 13/Cys 135 mutant exhibited a substantial decrease in stability and was not studied further. In contrast, the Cys 12/Cys 134 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 Cys 12/Cys 134 mutant was due to the substitution of Lys 12 and/or Pro 134 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 essential and similar or greater increases in stability can be realized with 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. 
     SUMMARY OF THE INVENTION 
     With the foregoing in mind, the present invention advantageously provides several purified polypeptides, the amino acid sequence of which consists of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. The purified polypeptides of the present invention exhibit from approximately fifteen to one thousand times more mitogenic activity than wild-type fibroblast growth factor 1 (FGF-1) in stimulating fibroblasts to proliferate. 
     In the present invention, it should be understood that the term “tissue” or “biological tissue” is employed to mean a collection of cells, which may be interconnected, and that perform a similar function within an organism; the tissue may be in vivo when in a living host or in vitro when the collection of cells is isolated in a laboratory culture. It is further to be understood that such a tissue within the meaning in the present invention will typically also have within it a certain number of connective cells known as fibroblasts. A fibroblast is a type of cell that synthesizes and maintains the extracellular matrix of many animal tissues. Fibroblasts provide a structural framework (stroma) for many tissues, and play a critical role in wound healing. They are the most common cells of connective tissue in animals. The main function of fibroblasts is to maintain the structural integrity of connective tissue by continuously secreting precursors of the extracellular matrix. Fibroblasts secrete the precursors of all the components of the extracellular matrix, primarily the ground substance and a variety of fibres. The composition of the extracellular matrix determines the physical properties of connective tissues. NIH 3T3 fibroblasts are a well established cell line which is representative of fibroblasts in general and are used experimentally as a type culture of human fibroblasts. 
     Since fibroblasts are responsible for synthesizing the components which make up connective tissue, they are known to be intimately involved in tissue repair, for example, in the healing of wounds. Accordingly, the artificially produced mutant polypeptides herein disclosed and which have been shown to have greatly enhanced mitogenic effects on fibroblasts, would be recognized by the skilled to be compounds applicable to treatment of tissues for promoting healing. The present invention, therefore, includes a method of treating fibroblasts, the method comprising contacting the fibroblasts with one or more of the mutant FGF-1 polypeptides herein disclosed. Moreover, the invention also includes a method of treating a biological tissue having fibroblasts, the method comprising contacting the biological tissue with a polypeptide consisting of one of the presently disclosed mutant FGF-1 sequences to thereby stimulate the fibroblasts. In the invention, a physiologically acceptable pharmaceutical composition could be formulated to contain the polypeptide. The pharmaceutical composition containing the polypeptide could be employed in a method of treatment by contacting the biological tissue with the pharmaceutical. Moreover, the skilled will also recognize that the polypeptides of the present invention or a composition containing one or more of the polypeptides could be borne on a medical device which would be used to contact the tissue so as to deposit some of the polypeptide thereon. For example, a medical device as simple as an elastic bandage strip, such as a BandAid® brand adhesive bandage strip, could be impregnated with the polypeptide or with a pharmaceutical composition containing the polypeptide. Applied to a cut, scrape or laceration, such a bandage would promote healing by depositing some of the polypeptide on the wound so as to thereby stimulate proliferation of the tissue fibroblasts. 
     A similar approach, albeit a bit more complex, would involve a medical device such as a cardiac catheter. The catheter could bear an amount of the polypeptide, either alone or in a pharmaceutical composition, so as to contact the vascular or cardiac tissue therewith to promote healing. Accordingly, the invention also includes a method of treating a biological tissue having fibroblasts, the method comprising contacting the biological tissue with a medical device bearing a physiologically acceptable pharmaceutical composition containing sufficient polypeptide of the sequences herein disclosed, to thereby stimulate the fibroblasts to proliferate. 
     The skilled will, no doubt, recognize that the range of medical devices which could be employed to bear a polypeptide of the present invention is quite broad. Any type of laparoscopic instrument, for example, could be coated with the present polypeptide or a composition containing the polypeptide. Instruments for arthroscopic surgery, for example, surgery involving the ligaments of the knee, would also benefit from the present invention. Similarly, endoscopes could either be coated with the polypeptide or a composition containing the polypeptide, or might be specially adapted to dispense the polypeptide during the endoscopy, for example, directly on a site where an intestinal polyp has just been excised. In short, the skilled will appreciate that any type of medical instrument or medical device which will come into contact with tissue which is damaged, or which will be damaged by the instrument or device, could be coated with the mutant polypeptides herein disclosed in order to stimulate fibroblasts to proliferate and, thus, to repair the damage. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Some of the features, advantages, and benefits of the present invention having been stated, others will become apparent as the description proceeds when taken in conjunction with the accompanying drawings, presented for solely for exemplary purposes and not with intent to limit the invention thereto, and in which: 
         FIG. 1  is a ribbon diagram of FGF-1, 18  showing the location of positions Lys 12 and Pro 134; the view on the right is looking down the β-barrel axis; also shown are the turn regions (residues 49-52 and 90-93) related to the N- and C-termini by the pseudo-three-fold axis of symmetry inherent in the β-trefoil architecture; 
         FIG. 2  is a relaxed stereo diagram of the local structure of FGF-1 in the region of positions Lys 12 and Pro 134 and including the hydrogen-bonding network; also shown are two small solvent excluded cavities, detectable using a 1.2 Å radius probe; 
         FIG. 3  is a relaxed stereo diagram showing an overlay of the K12→C, K12→T, and K12→V x-ray structures with WT* (dark grey) in the region of the mutation site; the solvent excluded cavity adjacent to position 12 is filled with each mutation; 
         FIG. 4  shows a relaxed stereo diagram showing an overlay of the P134-C x-ray structure with WT* (dark grey) in the region of the mutation site. The solvent excluded cavity adjacent to position 12 is no longer detectable due to the rotation of the Leu14 side chain; 
         FIG. 5  is a relaxed stereo diagram showing an overlay of the K12→V/N95→V x-ray structure with WT* (dark grey) in the region of the mutation site; the solvent excluded cavities adjacent to position 12 and 134 are no longer detectable due to the position 12 mutation and the adjustment of Pro 134 in response to the position 95 mutation; 
         FIG. 6  shows folding and unfolding kinetics “chevron plot” for WT* (•), K12→V(Δ), P134→V(□), K12→V/N95→V(∘), Lys12→Val/Leu46→Val/Glu87→Val/Asn95→Val/Pro134→Val (∇); and 
         FIG. 7  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). 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     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. 
     Materials and Methods 
     Mutagenesis and Expression 
     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. 
     Isothermal Equilibrium Denaturation 
     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 0.1 M 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   ]         )       /   R     ⁢           ⁢   T             1   +     ⅇ     -     (       Δ   ⁢           ⁢   G   ⁢           ⁢   0     +         m   ⁡     [   D   ]       /   R     ⁢           ⁢   T                         (   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) 
       FIG. 7  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  FIG. 7  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)). 
     Folding Kinetics Measurements 
     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 
     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. 
     Folding and Unfolding Kinetics Analysis 
     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 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:
 
 l ( t )= A exp(− kt )+ C   (3)
 
     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, 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 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 0 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    
     Mitogenic Assay 
     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. 
     Results 
     Mutant Protein Purification 
     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). 
     Isothermal Equilibrium Denaturation 
     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           Lys12→Cys/Pro134→Cys   36.2 ± 0.5   19.6 ± 0.3   1.84 ± 0.01   −14.5       Lys12→Cys/Pro134→Cys (oxidized)   30.5 ± 0.7   18.3 ± 0.4   1.66 ± 0.01   −10.6       Lys12→Cys   29.2 ± 0.7   19.4 ± 0.6   1.50 ± 0.01   −7.8       Lys12→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       Lys12→Val/Pro134→Val   37.6 ± 0.6   18.5 ± 0.3   2.03 ± 0.01   −17.7       Lys12→Val/Asn95→Val   34.1 ± 1.0   18.3 ± 0.6   1.86 ± 0.02   −14.4       Leu46→Val/Pro134→Val   27.7 ± 0.7   19.4 ± 0.5   1.43 ± 0.02   −6.4       Glu87→Val/Pro134→Val   27.7 ± 0.9   18.8 ± 0.6   1.47 ± 0.02   −7.0       Leu46→Val/Glu87→Val/Pro134→Val   28.8 ± 1.3   18.7 ± 0.8   1.54 ± 0.02   −8.4       Lys12→Val/Leu46→Val/Glu87→Val/   40.6 ± 1.3   16.6 ± 0.6   2.44 ± 0.02   −24.1       Asn95→Val/Pro134→Val               *Reference protein for all studies is FGF-1 Cys117→Val mutant (see text).         a ÄG value extrapolated to 0M denaturant.         b ΔΔG = (C mWT* −  C m mutant)(mWT*  +  mmutant )/2 as described by Pace and Scholtz 25 ;       a negative value of ÄÄG 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 f3-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. 
     Folding and Unfolding Kinetics 
     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 r     m r     k a     M u         Mutant   (s −1 )   (M −1 )   (1 × 10 −3  s −1)     (M −1 )                    WT*   3.75   −6.61   0.71   0.47       Lys12→Cys   15.0   −6.38   0.27   0.69       Lys12→Thr   28.8   −5.94   0.62   0.49       Lys12→Val   36.9   −6.08   0.56   0.55       Pro134→Cys   29.1   −6.84   0.41   0.56       Pro134→Thr   7.15   −6.17   0.43   0.52       Pro134→Val   6.12   −4.96   0.10   0.86       Lys12→Val/   125   −6.06   0.09   0.86       Pro134→Val       Lys12→Val/   161   −6.88   0.15   1.06       Asn95→Val       Leu46→Val/   8.04   −6.18   0.53   0.61       Pro134→Val       Leu46→Val/   25.4   −6.49   0.70   0.57       Glu87→Val/       Pro134→Val       Lys12→Val/   1,350   −5.51   0.11   1.10       Leu46→Val/       Glu87→Val/       Asn95→Val/       Pro134→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 Lys12→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. 
     X-Ray Structures 
     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. 
     Mitogenic Activity 
     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. 
     Discussion 
     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 ( FIG. 2 ) 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 ( FIG. 3 ). Both the Thr and Val mutations at position 12 adopt rotamer angles that orient a side chain ã 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+) ( FIG. 4 ). 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 ä 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 ( FIG. 5 ). 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 95 and 134, but not position 12, are unique in increasing the unfolding kinetics “m value” (i.e. cooperativity of unfolding;  FIG. 6 ) 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. 9  Thus, heparin functionality fell into the category of a demonstrable “function/stability trade-off” 19 -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 position 12, 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: 
     Lys12Cys/P134Thr, also designated herein as SEQ ID NO:15; 
     Lys12Cys/P134Val, also designated herein as SEQ ID NO:16; 
     Lys12Thr/P134Cys, also designated herein as SEQ ID NO:17; 
     Lys12Thr/P134Thr, also designated herein as SEQ ID NO:18; 
     Lys12Thr/P134Val, also designated herein as SEQ ID NO:19; 
     Lys12Val/P134Cys, also designated herein as SEQ ID NO:20; and 
     Lys12Val/P134Thr, also designated herein as SEQ ID NO:21; 
     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. 
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The β-trefoil protein human fibroblast growth factor-1 (FGF-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.