Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
1 To whom correspondence should be addressed at: NovImmune SA, 64 avenue de la Roseraie, CH-1211 Geneva 4, Switzerland. e-mail: nfischer{at}novimmune.com
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Abstract |
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Keywords: in vitro protein evolution/novel protein folds/phage display/proteolytic selection
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Introduction |
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While protein domains are 100250 residues long (Chothia et al., 2003
), typical exons encode polypeptides of about 40 amino acid residues on average (Fedorova and Fedorov, 2003
). This suggests that the assembly of several domains in one polypeptide must have involved the shuffling of either large exons or groups of exons (Blake, 1983
; Murzin et al., 1995
). Correspondingly, it has been proposed that individual protein domains may have arisen by the recombination of smaller exons (Blake, 1983
). Some support for this proposal comes from studies in experimental evolution, in which novel proteins were derived by random combination of non-homologous polypeptide segments of about 40 amino acid residues. In these studies, bait DNA encoding the N-terminal half of a ß-barrel domain was fused with fragmented genomic Escherichia coli DNA and cloned for display on filamentous bacteriophage. Phage displaying folded polypeptides were selected by proteolysis; in most cases the protease-resistant polypeptides comprised genomic fragments in their natural reading frames. Furthermore, only those comprising natural reading frames were soluble when expressed in the cytoplasm of E.coli (Riechmann and Winter, 2000
).
Here we have undertaken further studies and explored the use of a bait DNA encoding a designed polypeptide of a different architecture. This bait encoded two ß-strands of the variable domain of a human immunoglobulin V chain joined by a glycine spacer to a third strand in an attempt to retain a three-stranded ß-sheet architecture. The bait DNA was combined with random human cDNA and the resulting chimaeric proteins were displayed on filamentous bacteriophage. Stably folded domains were enriched by proteolysis, the selected chimaeric domains expressed as soluble proteins and their biochemical properties analysed.
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Materials and methods |
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We used for cloning the pHEN-D-TAG phagemid vector, modified from pHEN1, which contains the H102A mutant of barnase between the pelB leader peptide and the gene for protein 3 (p3) of fd phage (Hoogenboom et al., 1991; Meiering et al., 1992
). DNA fragments encoding residues 126 and 6877 of a human immunoglobulin V
light chain (012/DPK9) (Cox et al., 1994
) were amplified by polymerase chain reaction (PCR). Two different sets of primers encoding either two or three glycines were used, so that assembly of the PCR products yielded two different linker lengths in the final construct. During amplification, the mutation C23S was introduced in the N-terminal part of the V
domain. The polypeptides encoded by the resulting DNA fragments are LQDIQMTQSP SSLSASVGDR VTITSRASGG GGTDFTLTIS SGAQ and LQDIQMTQSP SSLSASVGDR VTITSRASGG GTDFTLTISS GAQ (sequences originating from the V
domain are underlined). The N-terminal LQ and C-terminal GAQ residues are partially encoded by PstI and SacI restriction sites present at the 5'- and 3'-ends of the PCR products, respectively. These PCR products were cloned between the PstI and SacI sites of pHEN-D-TAG to obtain the pV
-bait2 and pV
-bait3 vectors. In these constructs, the barnase/V
-bait fragments are out of frame relative to the gene of p3. The control vectors pDPK-9 and pV
-Bait were obtained by amplifying the whole V
DPK-9 domain or the V
-Bait (containing three glycines) with appropriate primers so that after cloning into pHEN-D-TAG a continuous open reading frame between barnase and p3 is restored. Restoration of the reading frame introduces an opal stop codon before p3. The presence of this stop codon reduces expression levels and presumably toxicity effects related to the expression of p3 fusions but allowing sufficient display on phage (Riechmann and Winter, 2000
).
Human mRNA isolated from HeLa cells was used for a first strand synthesis reaction with oligo-dT primers. Single-stranded DNA was amplified in 30 cycles of random PCR using 20 pmol/ml oligonucleotide SN6 (5'-GAG CCT GCA GAG CTC CGG NNN NNN-3') and an annealing temperature of 30°C. PCR products were amplified for another 30 cycles after adding 500 pmol/ml oligonucleotide NOARG (5'-CGT GCG AGC CTG CAG AGC TCA GG-3') and using a temperature of 52°C for annealing. Products between 150 and 250 bp were excised from an agarose gel and reamplified (20 cycles) with oligonucleotide NOARG. The PCR products were digested with SacI for cloning into SacI digested and dephosphorylated pV-bait2 and pV
-bait3 vectors. The ligation products were electroporated into E.coli strain TG1 and phages were rescued with the trypsin-sensitive KM13 helper phage (Kristensen and Winter, 1998
).
Proteolytic selections and screenings
Approximately 1011 colony-forming units were incubated for 10 min at 10°C with 200 nM TCPK-treated trypsin (Sigma) in TBS-Ca buffer (25 mM Tris, 137 mM NaCl, 1 mM CaCl2, pH 7.4). Phages were then mixed with one volume of 4% Marvel-PBS and transferred to a streptavidin-coated microtitre plate with bound biotinylated C40A/C82A mutant of barstar (Hartley, 1993; Lubienski et al., 1993
). Resistant phages were captured for 1 h at room temperature via the N-terminal barnase tag. Wells were washed 20 times with PBS, 5 min with 50 mM DTT in PBS to wash proteolysed phages remaining bound via disulfide bridges, followed by five additional PBS washes. Bound phages were eluted with 0.1 M glycine, pH 2.2, for 5 min and neutralized with one-tenth volume of 1 M Tris, pH 8. Eluted phages were used to infect TG1 cells for propagation.
Phage supernatants were screened by proteolysis in situ after capture on barstar-coated wells. Washes were performed as described above and bound phages were detected in ELISA with an anti-M13 phage antibodyhorseradish peroxidase conjugate (Amersham). For proteolysis in solution, 1010 purified phages were treated with trypsin for 5 min at different temperatures, before inactivation of the protease with Pefablock (Roche) and capture on immobilized barstar.
Protein expression, purification and analysis
DNA fragments encoding chimaeric proteins were amplified by PCR with appropiate oligonucleotides and recloned into the bacterial expression vector pQE30 encoding an N-terminal hexahistine tag (Qiagen) using HindIII and BamHI restriction sites. During amplification the opal stop codon was converted into TGG. Soluble chimaeric proteins consequently have the N-terminal tag MRGHH HHHHG SQ followed by the chimaeric protein followed by the C-terminal tag WAKLN.
For expression, exponentially growing bacteria (0.5 l cultures in 2 l conical flasks) were induced for 4 h at 30°C. Proteins were purified from the soluble fraction of the bacterial cytoplasm using the B-Per bacterial protein extraction reagent (Pierce), according to the manufacturers instructions and nitrilotriacetic acid (NTA) agarose (Quiagen). Alternatively, the 2a6 protein was prepared by resuspending the induced bacterial pellet in 10 mM Tris, pH 8.0, followed by heating at 90°C for 1015 min. The suspension was allowed to cool and centrifuged. Refolded 2a6 could then be purified from the supernatant using NTA agarose. No difference was detected between 2a6 samples prepared by either method. The protein was further purified by gel filtration on a Superdex-75 column (Amersham). The expected molecular weight of the protein was confirmed by surface-enhanced laser desorption/ionization (SELDI) (Ciphergen).
Soluble 2a6 protein was cross-linked in the presence of 1% glutaraldehyde for 2 min at 25°C using 5, 10 or 16 µM 2a6 monomer equivalents and analysed by SDSPAGE.
Biophysical analyses
Circular dichroism (CD) spectra were recorded with a Jasco J-720 spectropolarimeter and the temperature was adjusted with a Jasco PTC-348WI temperature controller. 2a6 dimer (12 µM monomer equivalents) in PBS was heated at rates of 60 or 80°C/h with very similar results and its ellipticity during denaturation was followed at 225 nm. Data were fitted to a two-state model between unfolded monomer and folded dimer using equation 17 in Mateu and Fersht (1998). The midpoint of thermal unfolding (Tm) and the enthalpy change for unfolding (
H) were inferred from the thermodenaturation curve and a
Cp of 12 cal mol1 K1 per residue (Pace et al., 1989
) was assumed (
Cp = 2808 cal mol1 K1 for the 2a6 dimer).
Fluorescence measurements were made using a Hitachi f-4500 spectrofluorimeter; 1.5 µM monomer equivalents of 2a6 were equilibrated in different concentrations of urea in PBS for at least 16 h at 15°C and its fluorescence emission was followed at 360 nm (excitation at 280 nm). Data were fitted to a two-state model for a direct transition between an unfolded monomer (Mu) and a folded dimer (Df) using equation 17 in Mateu and Fersht (1998).
Folding and unfolding of 2a6 (2 µM monomer equivalents) at 25°C were measured using a stopped-flow fluorimeter (Applied Photophysics SX17). Excitation was at 280 nm and emission was measured with a 360 nm cut-off filter.
For refolding experiments, 56 µM 2a6 was denatured in 4 M urea in PBS, pH 7, for 1 h at 25°C. Samples were then diluted to 22 µM 2a6 in 2.2 M urea in PBS to allow a stopped-flow analysis at lower denaturant concentrations. For refolding, denatured 2a6 was mixed with a 10-fold excess of different concentrations of urea (00.9 M) in PBS. The fluorescence decay of four traces was averaged and fitted to an equation combining a first-order and a second-order rate:
F(t) = Ffinal + A1exp(k1t) + A2/(Ptk2t + 1)
where F(t) is the time-dependent fluorescence signal, Ffinal the fluorescence signal at infinite time, A1 and A2 the changes in fluorescence signal due to the first and second reactions, k1 and k2 the rate constants for the first and second reactions, Pt the concentration of 2a6 monomer equivalents and t the time. Curve fitting to equations for a single first-order, a single second-order rate or two first-order rate reactions was unsatisfactory.
For unfolding experiments, 22 µM 2a6 dimer in PBS was mixed with a 10-fold excess of different concentrations of urea (0.54.5 M) in PBS. The fluorescence increase of four traces was averaged and fitted to an equation comprising two first-order rates:
F(t) = Ffinal + A1exp(k1t) + A2exp(k2t)
where F(t) is the time-dependent fluorescence signal, Ffinal the fluorescence signal at infinite time, A1 and A2 the changes in fluorescence signal due to the first and second reactions, k1 and k2 the rate constants for the first and second reactions and t the time. Curve fitting to an equation for a single first-order rate reaction was unsatisfactory.
One-dimensional proton nuclear magnetic resonance (1D 1H NMR) experiments were performed on a Bruker AMX-500 instrument with protein at 300 µM monomer equivalents in 20 mM phosphate, 0.1 M NaCl, pH 6.2, in 93% H2O7% D2O.
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Results |
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The two N-terminal ß-strands (residues 126, with a mutation Cys23Ser) and the seventh ß-strand of an immunoglobulin V chain (residues 6877), which comprise contiguous strands of a ß-sheet, were fused genetically through either two or three glycine residues. This polypeptide bait was cloned into a phagemid vector as a C-terminal fusion to barnase (used as an N-terminal affinity tag) followed by the minor phage coat protein p3 and thereby displayed on bacteriophage. The bait proved to be trypsin sensitive, suggesting that it was not folded (Kristensen and Winter, 1998
; Sieber et al., 1998
; Finucane and Woolfson, 1999
; Riechmann and Winter, 2000
; Martin et al., 2001
). The bait was then fused at its C-terminus with polypeptide fragments encoded by randomly amplified human cDNA of around 150250 bp to create a repertoire of 1.3x108 clones and selected by proteolysis with trypsin followed by capture on immobilized barstar via the N-terminal barnase tag. Selected phages were eluted at acidic pH and propagated in bacteria.
After two rounds of selection, phages with deletions started to dominate the repertoire (as analysed by PCR) and cDNA inserts of more than about 200 bp were PCR amplified from the population of phages and recloned into the phagemid vector for a third and final round of selection. Monoclonal phages isolated after the second and third rounds of selection were bound to immobilized barstar and proteolysed in situ with trypsin. Proteolytic resistance was assessed by detection of the phages remaining bound to the plate by ELISA. The cDNA inserts from 16 clones retaining at least 75% activity after proteolysis were sequenced and revealed seven different sequences, all of which were identified in the human genome. Three inserts (1c6, 3h8, 2f2) originated from coding sequences in the reading frame of the parent gene, three other inserts (1e4, 2a6, 1b5) were from antisense strands and one insert (3c12) corresponded to a 3' untranslated region (Table I). All of the clones contained the longer version of the linker (three glycines). When treated with trypsin over a range of temperatures, five clones (1e4, 2a6, 1c6, 3c12, 3h8) proved more resistant than the others (Figure 1A); the 2a6 clone dominated the library after the third round of selection.
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DNA encoding these five protease-resistant chimaeric polypeptides was recloned into a bacterial cytoplasmic expression vector encoding an N-terminal hexahistidine tag. The proteins 2a6 and 3c12 remained soluble, whereas the other polypeptides formed insoluble inclusions bodies. 3c12 suffered significant proteolytic degradation during expression and/or purification. 2a6, however, could be purified (several milligrams from 1 l of shaker flask culture) both under native conditions and after heating of the lysates without detectable degradation.
We probed the stability and folded nature of the soluble 2a6 protein by its resistance to proteolysis with trypsin, chymotrypsin and thermolysin at increasing temperatures (Figure 1B). 2a6 is largely resistant to all three proteases despite the presence of numerous potential proteolytic cleavage sites in its sequence (Figure 1D). The 2a6 protein was further purified by size-exclusion chromatography on a calibrated Superdex column. Its apparent molecular weight of 26 kDa (MWcalc. = 12 382 Da) suggests that the predominant oligomeric state of 2a6 is a dimer. The dimeric nature of 2a6 was confirmed by cross-linking of purified 2a6 (with 5, 10 and 16 µM monomer equivalents) in PBS with 1% glutaraldehyde for 2 min at 25°C, as cross-linked dimers but no higher order oligomers were seen (Figure 1C).
Further evidence for the folded nature of 2a6 was obtained from the one-dimensional 1H NMR spectrum, which shows chemical shift dispersion of amide protons to values downfield of 9 p.p.m. and of methyl groups to values around 0 p.p.m. (not shown). Chemical shift dispersion is indicative of a folded state as it is due to the variety of magnetic microenvironments present in folded proteins (Wüthrich, 1986). The CD spectrum of 2a6 was also consistent with components of both
and ß structure (Figure 2A). An
-helical content of
10% was estimated using the ellipticity at 208 nm (Greenfield and Fasman, 1969
). This is consistent with the secondary structure consensus prediction (Combet et al., 2000
) for 2a6, which suggests 17% of helical structure (residues 6675, 105114) and 21% of ß-structure (residues 1416, 3036, 4550, 7779, 8690).
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The fact that soluble and folded 2a6 could be so readily isolated from the bacterial lysate after heating to 90°C prompted us to analyse its stability and folding properties in more detail. Thermal denaturation of 2a6 was followed by CD at 225 nm and was fully reversible with a sharp sigmoidal melting curve (consistent with a cooperative process) with a melting point of 39°C (Figure 2B) at a concentration of 12 µM 2a6 monomer equivalents. A conformational stability GMu/Df of 10.9 kcal mol1 at 298 K was calculated by fitting the equilibrium data to a two-state model for a direct transition between unfolded monomer (Mu) and folded dimer (Df). The enthalpy (
H) was 82 kcal mol1 and a heat capacity change of 12 cal mol1 K1 per residue was assumed.
The degree of 2a6 unfolding in different concentrations of urea was followed by fluorescence emission spectroscopy from the single tryptophan residue close to its C-terminus. The change in fluorescence emission between native and urea unfolded 2a6 suggests that W113 is located in a more hydrophobic environment when folded (Figure 2C), as the emission maximum of tryptophan is shifted to shorter wavelength (Schmid, 1989). The fitting of the obtained sigmoidal transition curve to a simple two-state model for a direct transition between unfolded monomer (Mu) and folded dimer (Df) yielded the urea concentration for 50% denatured protein, [urea]50% = 1.4 M,
GMu/Df = 11.8 kcal mol1 and an m value of 3.9 kcal mol1 M1 (Figure 2D).
Folding and unfolding rates of 2a6 in urea at 25°C were measured using stopped-flow fluorescence experiments (Figure 3). Two reaction rates were observed during folding and extrapolated to 0 M denaturant as k1 = 0.0408 s1 (m1 = 2.1 kcal mol1 M1) and k2 = 4.56 M1 s1 (m2 = 1.8 kcal mol1 M1). Two reaction rates were also observed during unfolding and were extrapolated to 0 M denaturant as k1 = 0.00975 s1 (m1 = 1.1 kcal mol1 M1) and k2 = 0.0353 s1 (m2 = 1.2 kcal mol1 M1). The rates k1 and k1 were determined using the curve-fitting equation for the Chevron plot (Figure 3C). The rates k2 and k2 were determined by linear extrapolation of lnkobs to 0 M denaturant (Figure 3D).
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The two unfolding rates can be put in a sequential order. During the unfolding, the faster rate (k2 = 0.0353 s1 when extrapolated to 0 M denaturant) must precede the slower rate (the extrapolated k1 = 0.00975 s1), otherwise only the rate-determining slower rate would be observable. One of these (first-order unfolding rates) must describe the reverse reaction of the one first-order reaction (k1) observed during refolding. This rate must be k1, because only the k1 rates (and not the k2 rates), which were observed at different urea concentrations, can be combined with the observed k1 rates in a Chevron plot (Figure 3C) using equation 18.6 in Fersht (1998):
lnkobs = ln{k1,waterexp(m1[urea]) + k1,waterexp(m1[urea])}
Hence the unfolding reaction described by k1 is the second step during unfolding and the reverse reaction of that described by k1. As the folding reaction described by k1 is first order (i.e. unimolecular), k1 must be the rate constant for the folding of the 2a6 monomer. Then the second-order refolding rate k2 must describe the second step during refolding (that is, the dimerization of the folded 2a6 monomers) with k2 describing the dissociation of this dimer during unfolding. Their values at 0 M denaturant were determined by linear extrapolation of the respective lnkobs (Figure 3D).
The conclusions regarding the folding and unfolding rates are summarized in the diagram for the proposed folding pathway of 2a6 (Figure 4). The attribution of the determined rate constants to the specific steps of the folding pathway makes it possible to use the ratio of forward and backward rate constants associated with a specific folding step to determine the free energy difference between the reactants and products of this step according to:
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for the G between unfolded and folded monomer and
G(Mf/Df) = RTln(k2/k2) = 9.7 kcal mol1
for the G between folded monomer and folded dimer.
Combination of these two G values [2
G(Mu/Mf) +
G(Mf/Df)] yields
G(Mu/Df) = 11.4 kcal mol1 between unfolded monomer and folded dimer. This number is in reasonable agreement with the
G(Mu/Df) determined from the equilibrium unfolding experiment (urea unfolding 11.8 kcal mol1; thermal unfolding 10.9 kcal mol1). Hence the very short-lived accumulation of the folded monomer during unfolding does not significantly influence the determination of the free folding energy in the equilibrium denaturation experiments, where this intermediate is presumed absent.
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Discussion |
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2a6 forms homodimers featuring cooperative and reversible unfolding during both thermal and chemical denaturation. Furthermore, the 2a6 dimer is largely resistant to proteolysis, both as a phage displayed fusion protein and in its soluble form. Other indicators for the folded nature of 2a6 are its CD spectrum, which is consistent with structural elements of both ß and structure, and its distinct oligomerization status as a dimer. Unfortunately, the NMR signals for 2a6 are too broad to allow the determination of its solution structure and we have so far been unable to obtain crystals of 2a6 to determine its X-ray structure. It is therefore unclear whether the designed ß-sheet structure of the bait is retained within 2a6. Indication of a helical structure from the CD data, which according to the secondary structure prediction is most likely located in its C-terminal half, furthermore makes it unlikely that 2a6 folds into an immunoglobulin-like domain. However, the biochemical and biophysical analyses show that this novel domain is fully folded and has hallmarks of native proteins which are not found in less compact, native-like structures defined as molten globules (DeGrado et al., 1999
).
The folding stability of 2a6 is 1112 kcal mol1. This value is typical for dimers of natural proteins.
G for the unfolding of the Arc repressor dimer (2x53 residues) is 10 kcal mol1 (Milla and Sauer, 1994
).
G of the leucine zipper peptide GCN-4 dimer (2x33 residues) is 10.5 kcal mol1 (Zitzewitz et al., 1995
) and that of the E2 DNA binding domain dimer (2x80 residues) of the human papillomavirus is 11 kcal mol1 (Mok et al., 1996
). Accordingly, the folding and unfolding rates of the 2a6 dimer (4.6x105 M1 s1 and 0.035 s1) are also in line with those seen in natural dimers (Jackson, 1998
). The unfolding rate of the 2a6 monomer (0.0098 s1) is also within normal values for natural monomeric proteins (Jackson, 1998
), whereas the monomer folding rate (0.041 s1) is rather slow, which is reflected in the very low stability of the transient 2a6 monomer (0.85 kcal mol1).
The favourable and native-like folding properties are reflected in the ability of 2a6 to refold readily from heat-denatured bacterial lysates during purification, apparently without aggregation. The refolding rates of 2a6 are also consistent with the absence of alternative, unproductive folding pathways. If present, such pathways often lead to transiently accumulating aggregates at lower denaturant concentrations and a non-linear behaviour of the folding arm of the Chevron plot (Figure 3) (Silow and Oliveberg, 1997).
The dimer formation of 2a6 may be related to our selection process. Two out of four stably folded chimaeric proteins, in which the N-terminal half of CspA was recombined with random DNA fragments of bacterial origin, were found to be multimeric [1c2 and 1b11 in Riechmann and Winter (2000), whose oligomerization status was wrongly described in the original reference]. While the use of a helper phage and the opal stop codon within gene 3 of the phagemid (see Materials and methods) must favour the expression of monomers in most phage, it does not exclude the presence of multimers in a proportion of the phage. Indeed, the fact that the fusions are to the multivalent phage coat protein p3 (Model and Russel, 1988
) may favour the selection of multimers over monomers in two respects. First, multimers are more likely to be stable and resist proteolysis; secondly, phage bearing protease-resistant multimers should be captured more readily owing to the greater avidity of binding of the multimeric barnase affinity tags.
A number of studies in the recent past have been aimed at generating novel proteins from partially or fully randomized polypeptide libraries, using different selection or screening strategies (Davidson and Sauer, 1994; Davidson et al., 1995
; Doi et al., 1997
, 1998
; Keefe and Szostak, 2001
). However, in most cases the selected polypeptides were insoluble or poorly structured when expressed on their own and had to be characterized in the presence of urea or guanidium chloride. We have shown that combinatorial shuffling of natural DNA fragments of human (this study) or bacterial (Riechmann and Winter, 2000
) origin can be successfully used to create folded protein domains. In our earlier work, the three proteins that were soluble when expressed in E.coli proved to combine natural reading frames (Riechmann and Winter, 2000
). Here we have shown that the protein 2a6 not only comprises a bait based on a designed ß-sheet but also an antisense read of a human gene. This suggests that non-coding DNA fragments could be mobilized in protein evolution through recombination events to generate soluble folded proteins directly. This would be expected to increase the evolutionary potential of genomes and complements exon shuffling as a productive way of recombining segments of coding DNA (Kolkman and Stemmer, 2001
).
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Acknowledgements |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blake,C. (1983) Nature, 306, 535537.[ISI][Medline]
Bogarad,L.D. and Deem,M.W. (1999) Proc. Natl Acad. Sci. USA, 96, 25912595.
Chothia,C., Gough,J., Vogel,C. and Teichmann,S.A. (2003) Science, 300, 17011703.
Combet,C., Blanchet,C., Geourjon,C. and Deleage,G. (2000) Trends Biochem. Sci., 25, 147150.[CrossRef][ISI][Medline]
Cox,J.P., Tomlinson,I.M. and Winter,G. (1994) Eur. J. Immunol., 24, 827836.[ISI][Medline]
Davidson,A.R. and Sauer,R.T. (1994) Proc. Natl Acad. Sci. USA, 91, 21462150.[Abstract]
Davidson,A.R., Lumb,K.J. and Sauer,R.T. (1995) Nat. Struct. Biol., 2, 856864.[ISI][Medline]
DeGrado,W.F., Summa,C.M., Pavone,V., Nastri,F. and Lombardi,A. (1999) Annu. Rev. Biochem., 68, 779819.[CrossRef][ISI][Medline]
Doi,N., Itaya,M., Yomo,T., Tokura,S. and Yanagawa,H. (1997) FEBS Lett., 402, 177180.[CrossRef][ISI][Medline]
Doi,N., Yomo,T., Itaya,M. and Yanagawa,H. (1998) FEBS Lett., 427, 5154.[CrossRef][ISI][Medline]
Fedorova,L. and Fedorov,A. (2003) Genetica, 118, 123131.[CrossRef][ISI][Medline]
Fersht,A.R. (ed.) (1998) Structure and Mechanism in Protein Science. Freeman, San Francisco, pp. 540572.
Finucane,M.D. and Woolfson,D.N. (1999) Biochemistry, 38, 1161311623.[CrossRef][ISI][Medline]
Greenfield,N. and Fasman,G.D. (1969) Biochemistry, 8, 41084116.[ISI][Medline]
Hartley,R.W. (1993) Biochemistry, 32, 59785984.[ISI][Medline]
Hoogenboom,H.R., Griffiths,A.D., Johnson,K.S., Chiswell,D.J., Hudson,P. and Winter,G. (1991) Nucleic Acids Res., 19, 41334137.[Abstract]
Hosszu,L.L., Craven,C.J., Parker,M.J., Lorch,M., Spencer,J., Clarke,A.R. and Waltho,J.P. (1997) Nat. Struct. Biol., 4, 801804.[ISI][Medline]
Jackson,S.E. (1998) Fold. Des., 3, R81R91.[ISI][Medline]
Keefe,A.D. and Szostak,J.W. (2001) Nature, 410, 715718.[CrossRef][ISI][Medline]
Kolkman,J.A. and Stemmer,W.P. (2001) Nat. Biotechnol., 19, 423428.[CrossRef][ISI][Medline]
Kristensen,P. and Winter,G. (1998) Fold. Des., 3, 321328.[ISI][Medline]
Lubienski,M.J., Bycroft,M., Jones,D.N. and Fersht,A.R. (1993) FEBS Lett., 332, 8187.[CrossRef][ISI][Medline]
Martin,A., Sieber,V. and Schmid,F.X. (2001) J. Mol. Biol., 309, 717726.[CrossRef][ISI][Medline]
Mateu,M.G. and Fersht,A.R. (1998) EMBO J., 17, 27482758.
Mayr,E.M., Jaenicke,R. and Glockshuber,R. (1997) J. Mol. Biol., 269, 260269.[CrossRef][ISI][Medline]
Meiering,E.M., Serrano,L. and Fersht,A.R. (1992) J. Mol. Biol., 225, 585589.[ISI][Medline]
Milla,M.E. and Sauer,R.T. (1994) Biochemistry, 33, 11251133.[ISI][Medline]
Model,P. and Russel,M. (1988) In Calender,R. (ed.), The Bacteriophages. Plenum Press, New York, pp. 375456.
Mok,Y.-K., De Prat Gay,G., Butler,P.J. and Bycroft,M. (1996) Protein Sci., 5, 310319.
Murzin,A.G., Brenner,S.E., Hubbard,T. and Chothia,C. (1995) J. Mol. Biol., 247, 536540.[CrossRef][ISI][Medline]
Pace,C.N., Shirley,B.A. and Thomson,J.A. (1989) In Creighton,T.E. (ed.), Protein Structure, a Practical Approach. IRL Press, Oxford, pp. 311330.
Riechmann,L. and Winter,G. (2000) Proc. Natl Acad. Sci. USA, 97, 1006810073.
Schmid,F. (1989) In Creighton,T.E. (ed.), Protein Structure, a Practical Approach. IRL Press, Oxford, pp. 251285.
Sieber,V., Pluckthun,A. and Schmid,F.X. (1998) Nat. Biotechnol., 16, 955960.[ISI][Medline]
Silow,M., Oliveberg,M. (1997) Proc. Natl Acad. Sci. USA 94, 60846086.
Wüthrich,K. (1986) In Wüthrich,K. (ed.) NMR of Proteins and Nucleic Acids. Wiley, New York, pp. 2639.
Zitzewitz,J.A., Bilsel,O., Luo,J.B., Jones,B.E. and Matthews,C.R. (1995) Biochemistry, 34, 1281212819.[ISI][Medline]
Received October 2, 2003; accepted October 14, 2003 Edited by Alan Fersht