MBE Advance Access originally published online on March 18, 2008
Molecular Biology and Evolution 2008 25(6):1113-1119; doi:10.1093/molbev/msn063
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Research Articles |
Emergence of Polyproline II-Like Structure at Early Stages of Experimental Evolution from Random Polypeptides
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* Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan
Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Suita, Osaka, Japan
Graduate School of Frontier Science, Osaka University, Suita, Osaka, Japan
Complex Systems Biology Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Suita, Osaka, Japan
E-mail: yomo{at}ist.osaka-u.ac.jp
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Abstract |
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To examine whether a primordial functional protein at the early stages of evolution has structural features, we carried out experimental evolution consisting of 25 cycles (generations) of mutation and selection toward DNA-binding function using a random-sequence polypeptide of 139 amino acid residues with no secondary structure as the initial sequence. In each generation, 16 clones were sampled arbitrarily for sequence analysis, and a phylogenetic tree was constructed. Polypeptide evolution proceeded from the initial point on branch I in 2 main directions of branches II and III. The initial and 2 evolved polypeptides (one at the 24th generation on branch III and the other at the 25th generation on branch II) were purified to examine their functional and structural properties. Although binding of the initial polypeptide to the target DNA was not detected by surface plasmon resonance measurements, the 2 evolved polypeptides bound to the DNA with dissociation constants of 1.6 and 1.0 µM, respectively, indicating an increase in affinity during the experimental evolution. Circular dichroism spectra of the evolved polypeptides, but not of the initial polypeptide, showed features characteristic of the polyproline II (PPII)–like structure, a left-handed helical structure commonly found in natural proteins, suggesting that the structure emerged through the experimental evolution. The same structural feature was found in another experimental evolution toward catalytic activity. These results demonstrate that the PPII-like structure is one of the common features that could have appeared in the early evolutionary stages of primordial functional protein.
Key Words: experimental evolution • early evolution • random polypeptide • polyproline II • DNA binding
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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In the early stages of protein evolution, the first functional proteins presumably originated from arbitrary polypeptides with random sequences. As the frequency of functional polypeptides in a random-sequence library was estimated experimentally to be 10–11 (Keefe and Szostak 2001
), a similar probability is expected if such innate functional polypeptides emerged suddenly at the early stages of evolution (Keefe and Szostak 2001). On the other hand, most trials of protein evolution unavoidably start with very low or negligible levels of function, as observed for random polypeptides (Prijambada et al. 1996; Yamauchi et al. 1998). Therefore, the evolvability of arbitrarily chosen random polypeptides was examined and demonstrated experimentally through cycles of mutation and selection toward several biological functions (Yamauchi et al. 2002; Hayashi et al. 2003; Ito et al. 2004; Nakashima et al. 2007).
In the latter case, assuming that the global structures of proteins coevolve gradually with their function, as suggested theoretically (Saito et al. 1997; Yomo et al. 1999), such primordial functional proteins obtained through experimental evolution may show traces of structural features. However, it is not known whether primordial functional proteins at the early stages of evolution already show some structural characteristics. Here, we report the emergence of a polyproline II (PPII)–like structure when an arbitrarily chosen soluble random polypeptide, RP3-42 (Prijambada et al. 1996), was subjected to experimental evolution through cycles of mutation and selection based on binding affinity to DNA. In addition, a similar structure was found in a primordial functional protein obtained previously (Yamauchi et al. 2002) at an early stage of experimental evolution based on another biological function. The PPII-like structure, which is not necessarily composed of proline residues, is a left-handed helical structure with 3 residues per turn, and is commonly found in natural proteins (Makarov et al. 1992; Adzhubei and Sternberg 1993; Sreerama and Woody 1994; Bochicchio and Tamburro 2002; Hicks and Hsu 2004; Cubellis et al. 2005). These experimental results support the theoretical proposition that structures coevolve gradually with function (Saito et al. 1997; Yomo et al. 1999), which is also a scenario proposed for protein diversification (James and Tawfik 2003), at the initial stages of protein evolution. The emergence of PPII-like structures by different types of experimental evolution suggests that PPII is one of the common structures that could have appeared in the early evolutionary stages of primordial functional protein.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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Bacterial Strains, Phage, Plasmids, and Oligonucleotides
Escherichia coli strains TG1 (supE, hsd
5, thi, (lac-proAB)/F’[traD36, proAB+, lacIq, lacZ, M15]) used for phage display and BL21(DE3) (F–, ompT, hsdSB(rB– mB–), gal, dcm (DE3)) used for protein expression were purchased from Amersham Biosciences (Piscataway, NJ) and Novagen (San Diego, CA), respectively. The phage clone displaying a random-sequence polypeptide, RP3-42, was prepared as described previously (Nakashima et al. 2000). The phagemid pCANSS, a derivative of pCANTAB5E (Amersham Biosciences), was prepared previously (Nakashima et al. 2000) and used for phage display. The plasmid pET21aSH, a derivative of pET21a(+) (Novagen), was prepared previously (Yamauchi et al. 2002) and used for expressing proteins with a C-terminal His6-tag. Oligonucleotides were purchased from Sigma Genosys Japan (Hokkaido, Japan).
Phage Preparation
Phage were prepared as described previously (Yamauchi et al. 2002) except that they were suspended in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)–buffered saline (HBS) (10 mM HEPES–NaOH, 50 mM NaCl, 1 mM MgCl2, pH 7.5) supplemented with 8% (v/v) NEB restriction enzyme diluent buffer A (10 mM Tris–HCl, 50 mM KCl, 0.1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol [DTT], 0.2 µg/ml bovine serum albumin, 50% (v/v) glycerol, pH 7.4).
Derivation of Phage Library Displaying Derivatives of RP3-42
The genes for RP3-42 and its derivatives on phagemids were amplified by mutagenic polymerase chain reaction (PCR) under the conditions of low mutational bias (deoxyguanosine triphosphate and deoxyadenosine triphosphate concentrations were each 0.2 mM) (Cadwell and Joyce 1992) using Tth DNA polymerase (Toyobo, Osaka, Japan) (Arakawa et al. 1996) and the primers, 5'-ATCCTCGCAACTGCGGCCCACGTGGCCATGGCTAGCATGACTGGTGGACAGCAAATGGGT-3' and 5'-AGTTTAGGCCACAGAGGCCTGGATCGCGAGATCTGTCGACTC-3'. The SfiI fragment from the PCR products was ligated into pCANSS, and E. coli TG1 cells were transformed with the ligated DNA by electroporation. A phage library was prepared from the phagemid library, the size of which was maintained at about 2 x 106, as described above. The mutation rate was about 0.01 per nucleotide site.
In Vitro Selection
Derivatives of a random polypeptide, RP3-42, displayed on the surface of M13 phage as a fusion protein with the pIII coat protein were selected based on their binding ability with double-stranded target DNA, including one of the artificial sequences that can be recognized by Zif268 reported by Choo et al. (1997) and an NcoI recognition site. The design and preparation of the target DNA were almost the same as those described previously (Nakashima et al. 2007). The sequence of the biotinylated oligonucleotide was biotin-5'-GACGTACCATGGAATTCGTACGCTCACGCTAGTAGCCCTCGAGATGCAG-3', where the underlined bases indicate the NcoI recognition site and the bases in bold type indicate the zinc-finger recognition site. Selection of phage-displayed polypeptides capable of binding to the target DNA was performed at 20 °C on a Biacore 2000 system (Biacore AB, Uppsala, Sweden). The Biacore instrument and a Sensor Chip SA (streptavidin) were equilibrated with HBS supplement with Tween 20 (10 mM HEPES–NaOH, 50 mM NaCl, 1 mM MgCl2, 0.005% Tween 20, pH 7.5) supplemented with 8% (v/v) NEB restriction enzyme diluent buffer A. The sensor chip was conditioned with NaCl/NaOH solution in accordance with the manufacturer's instructions. The biotinylated target DNA (1 µM) dissolved in 10 mM HEPES–NaOH (pH 7.5) containing 0.5 M NaCl and 1 mM MgCl2 was injected into a flow cell at a flow rate of 2 µl/min for 30 min. As a result, an increase of about 2,500 resonance units (RU) corresponding to the immobilization of 0.1 pmol of the target DNA on the sensor chip was observed. Then, aliquots of 4 x 1013/ml of the phage displaying polypeptides were injected into the flow cell at a flow rate of 1 µl/min for 30 min. After injection, the sensor chip was undocked from the Biacore instrument keeping the sensor surface wet, and background phage bound to the liquid delivery system of the instrument was washed out by 2 consecutive desorb commands followed by rinsing with water: first with 50 mM glycine (pH 9.5) and 0.5% sodium dodecyl sulfate (SDS) and second with 50 mM HCl and 70% ethanol; total washing time was about 1 h. No phage were detectable from the instrument after washing. The sensor chip was then docked again into the instrument and washed with HBST supplemented with 8% (v/v) NEB restriction enzyme diluent buffer A at a rate of 5 µl/min, and the flow-through buffer was recovered at constant time intervals using the inject (15 min) and recovery commands. At the last (fourth) recovery, HBST supplemented with 8% (v/v) NEB restriction enzyme diluent buffer A containing NcoI (final concentration, 4 units/µl; New England Biolabs, Ipswich, MA) was injected to recover the target DNA with bound phage by NcoI digestion. Completion of the digestion reaction was monitored on the Biacore sensorgram (Nilsson et al. 1995). The number of phage recovered was measured by determining the number of ampicillin-resistant colonies of phage-infected E. coli TG1 cells (Marks et al. 1991). All the colonies obtained from the last recovery were collected and stored at –80 °C.
Sequence Analysis
DNA sequencing was outsourced to Hitachi High-Technologies Corporation (Tokyo, Japan) or performed using a BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).
Expression and Purification of His-Tagged Polypeptides
The polypeptide gene on the phagemid carried by a selected phage was subcloned into the NheI/SfiI sites of pET21aSH. When the gene had an internal amber stop codon (TAG), which can survive in the supE strain of E. coli TG1, the codon was replaced by the glutamine codon (CAG) using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The polypeptide was expressed in E. coli BL21(DE3) cells harboring the hybrid plasmid by induction with isopropyl-β-D-thiogalactopyranoside. The induction conditions were 5 h at 30 °C for RP3-42H and TDP24-2H or 7 h at 20 °C for TDP25-4H. After induction, the cells were lysed in 0.1 M NaH2PO4, 10 mM Tris (pH 7.5) containing 0.5 M NaCl, 20 mM imidazole, and 8 M urea. The supernatant of the lysate was applied to a Ni-NTA column (Qiagen, Hilden, Germany) equilibrated with the same buffer on an AKTA prime FPLC system (Amersham Biosciences). The column was washed with the same buffer except that the imidazole concentration was increased to 30 mM, and the polypeptide was eluted with an imidazole gradient of 30–80 mM. The fractions showing a single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) were pooled and concentrated with a Vivaspin centrifugal concentrator (Vivascience, Hannover, Germany). The buffer solution was exchanged with HBS using a PD-10 desalting column (Amersham Biosciences), and the purified polypeptide was stored at 4 °C. All the purified polypeptides were confirmed to be homogeneous by SDS-PAGE and amino acid analysis (Suga et al. 1996). Protein concentration was also determined by amino acid analysis (Suga et al. 1996).
Gel Filtration
Gel filtration chromatography was performed at room temperature (20–25 °C) on a Superdex 200 PC 3.2/30 column (Amersham Biosciences) with a SMART system (Amersham Biosciences). The column was equilibrated with 10 mM HEPES–NaOH (pH 7.5) containing 150 mM NaCl and 1 mM MgCl2 or the same buffer supplemented with 8 M urea and 10 mM DTT for native or reducing denaturing conditions, respectively. The sample solutions were injected into the column, and the elution profiles were monitored by determining the absorbance at 280, 260, and 237 nm. The void and total volumes of the column were measured with blue dextran (2,000 kDa) and acetone, respectively. The following proteins in a molecular weight (MW) marker kit (Sigma, St. Louis, MO) were used as standards: sweet potato β-amylase, MW 200 kDa and Stokes radius (Rs) 50.4 Å; bovine serum albumin, MW 66 kDa and Rs 33.9 Å; bovine erythrocyte carbonic anhydrase, MW 29 kDa and Rs 23.6 Å; and horse heart cytochrome c, MW 12.4 kDa and Rs 17.0 Å. The Rs values of the polypeptides were calculated from the empirical linear relation between Rs and the inverse of elution volume (Uversky 1993).
Surface Plasmon Resonance
Surface plasmon resonance (SPR) measurements were performed at 20 °C in HBST supplemented with 8% (v/v) NEB restriction enzyme diluent buffer A at a flow rate of 30 µl/min on a Biacore 2000. The target DNA was immobilized on a Sensor Chip SA (about 400 RU). Association and dissociation phases were 180 and 300 s, respectively. After each measurement, the surface of the sensor chip was regenerated with pulses of 0.05% SDS solution. For binding experiments, samples with various polypeptide concentrations (2.5–100 nM) were injected. For solution affinity experiments, a sample polypeptide (final concentration 100 nM) was mixed with various concentrations (final 10–300 nM) of target DNA (without biotinylation) or nontarget DNA (double-stranded 5'-GTCGATGGTACCCCGTAGTAGGCCAATGGTAGCAATTGTGCACTAGCTG-3'), and the mixtures were incubated for 1 h at 20 °C before injection. The concentration of free polypeptide, Pfree, in each mixture was estimated using the standard curve obtained from the binding experiments. The dissociation constant, Kd, and the apparent site size, n, occupied by a bound polypeptide were estimated by nonlinear least square fitting to the equation:
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is the binding density, that is, the number of bound polypeptides per target DNA, N is the number of base pairs of the target DNA, (f) is the probability that a base pair of the target DNA is free, and (ff) is the conditional probability that the right-side base pair of a free base pair is also free. The equation was derived by extending the McGhee and von Hippel (1974) approach for noncooperative binding to a 1-dimensional infinite lattice into binding to a finite lattice by the following assumptions. At sufficiently low binding density, the probabilities (b1) that a residue is the first of an occupied site and (f) are assumed to be /N and 1 – n(/N), respectively, at every residue of the finite lattice, and hence (ff) becomes (f)/[(f)+(b1)]. In addition, the first residue of a potential binding site is assumed to lie within the first 
residues of the lattice. Hence, the total number of free binding sites per lattice is 
.
Circular Dichroism Spectroscopy
Circular dichroism (CD) spectra were recorded on a Jasco J-720 spectropolarimeter equipped with a Peltier temperature controller (PTC-348WI; Jasco, Tokyo, Japan). The spectra were scanned 8 times at a scan rate of 20 nm/min, using a response time of 0.25 s and a spectral band width of 1 nm, and the average of the 8 scans was recorded. The polypeptide concentration was in the range of 5–20 µM, and the light path length of the cell was 1 mm. Data points with high-tension voltage over 600 V were excluded from analysis. Appropriate background spectra were scanned and subtracted from polypeptide spectra.
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Results and Discussion |
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We started experimental evolution toward DNA-binding function using a random polypeptide, RP3-42, as the initial sequence. RP3-42 is a soluble random polypeptide of 139 amino acid residues with no secondary structure (Prijambada et al. 1996
; Yamauchi et al. 1998), which was used previously as an arbitrary sequence representing the initial material of an intended evolutionary study toward another biological function related to phage infectivity (Hayashi et al. 2003). As RP3-42 was shown to be evolvable in an intended direction (Hayashi et al. 2003), testing its evolvability in another direction (i.e., DNA binding) represents a test of the evolutional plasticity of an arbitrary sequence.
First, the RP3-42 gene on the phagemid was subjected to random mutagenesis, and the resulting library of mutant polypeptides displayed on phage was subjected to selection based on binding ability to a target DNA using a Biacore system. The system enables us to monitor the binding and elution processes and to control the selection processes under constant conditions. The target DNA contained a recognition site for the NcoI restriction enzyme, which was used to recover the bound phage as a complex with the target DNA (Nakashima et al. 2007). The selected phage formed the first-generation library. Further cycles of mutation and selection were carried out up to the 25th generation. The number of phage selected in each generation increased gradually, and the value for the 25th generation was about 2.2 x 103 times larger than that of the zeroth-generation phage displaying RP3-42 (fig. 1A). The increase in phage selection suggested an increase in the number of phage bound to the target DNA and hence an increase in the binding ability of the polypeptides displayed on the selected phage.
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In each generation, 16 clones were sampled arbitrarily, and the nucleotide sequences of the genes encoding the polypeptides were analyzed. The mean of the cumulative number of amino acid substitutions deduced from the nucleotide sequences increased concomitant with the increase in phage selection (fig. 1A). A phylogenetic tree was constructed based on the distances among the deduced amino acid sequences (fig. 1B). In the tree, the positions of each sequence are indicated by colored symbols, which correspond to the symbols used in figure 1A. The tree is composed of 3 main branches: I, II, and III (fig. 1B). The red symbol of the initial sequence (RP3-42) enclosed within a red circle is located on branch I, and sequences in generations 1–8 (orange to yellow) are also located on branch I forming a group. The positions of the sequences in generations 9–13 (yellow to green) are widely scattered and are located on branches I and II. After the 15th generation (cyan to magenta), no new sequences appeared on branch I, suggesting extinction of the large sequence group of the branch. On the other hand, branch III is formed only by the newer sequences appearing in and after the 14th generation (green to magenta). Overall, the evolution of the polypeptide sequence proceeded from the starting point on branch I in the 2 main directions of branches II and III. These results suggest that evolution under a constant selection pressure proceeds divergently but not isotropically; many sequences appear and become extinct, whereas several sequences survive on a limited number of groups forming small branches in different directions. That is, evolution seems to proceed not monodirectionally but in several directions, retaining the possibility of reaching several different endpoints.
To examine the functional and structural properties of the evolved polypeptides, we chose one clone from each of branches II and III (the magenta and purple circles in fig. 1B), and the polypeptides of these clones and also RP3-42 were expressed with a His6-tag in E. coli cells as soluble forms. The purified polypeptides were named TDP24-2H (selected at the 24th generation on branch III), TDP25-4H (selected at the 25th generation on branch II), and RP3-42H, respectively.
The MWs of RP3-42H, TDP24-2H, and TDP25-4H determined by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry were 15751, 15621, and 15290, respectively, indicating that one Met residue was missing in each sequence. The N-terminal Met residue was probably removed during expression, as reported previously (Hirel et al. 1989). The deduced sequences of TDP24-2H and TDP25-4H are shown in the legend to table 1.
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To estimate the compactness of RP3-42H, TDP24-2H, and TDP25-4H, their Stokes radii (Rs) were determined by gel filtration chromatography. In the native state, their values were similar and 1.1- to 1.2-fold greater than that expected for a globular protein of similar MW (table 1). In the denatured state with 8 M urea and 10 mM DTT, these values increased to almost the same as those expected for the denatured state of the calculated protein (table 1). These results suggest that these polypeptides in the native state are more compact than the denatured state but not as compact as native globular proteins.
The affinities of RP3-42H, TDP24-2H, and TDP25-4H to the target DNA were estimated by SPR analysis. Although the binding of the initial polypeptide, RP3-42H, to the target DNA was not detected, TDP24-2H and TDP25-4H bound to the DNA with dissociation constants, Kd, of 1.6 and 1.0 µM, respectively (table 1), indicating increases in DNA-binding affinity during the experimental evolution. It should be noted that they showed similar affinity to another DNA with the same composition but different sequence (data not shown), indicating that their DNA-binding ability was not specific to the target DNA sequence used for the selection. Although the level of the evolved function is still in a primitive state, the random polypeptide (RP3-42) was shown to be evolvable toward a DNA-binding protein. As the same random polypeptide was shown previously to be evolvable toward another biological function (Hayashi et al. 2003), these results demonstrate the evolutional plasticity of an arbitrary sequence in function.
To examine whether these polypeptides with a primitive function obtained through the experimental evolution have structural characteristics, CD spectra of TDP24-2H and TDP25-4H were measured together with RP3-42H at pH 7.5 and at various temperatures, as indicated in figure 2. The spectra of TDP24-2H and TDP25-4H were similar and showed characteristic features in that they each had a negative band at around 200 nm, and the trough deepened with decreases in temperature with a concomitant increase in a band around 220 nm forming an isodichroic point around 209 nm. As these features are characteristic of the PPII structure (Bochicchio and Tamburro 2002), the 2 polypeptides were suggested to contain a PPII-like structure. The PPII-like structure, not necessarily composed of proline residues, is a left-handed helical structure with 3 residues per turn and is commonly found in natural proteins (Makarov et al. 1992; Adzhubei and Sternberg 1993; Sreerama and Woody 1994; Bochicchio and Tamburro 2002; Hicks and Hsu 2004; Cubellis et al. 2005). The presence of the isodichroic point indicates that the polypeptides existed in an equilibrium state between PPII and other conformations. The results of singular value decomposition analysis (Henry and Hofrichter 1992; Johnson 1992) of the spectra suggested that the significant components of the conformations of the polypeptides were PPII and probably disordered conformations, corresponding to the second and the first basis spectrum, respectively (supplementary fig. S3, Supplementary Material online). The difference spectra generated by subtracting higher temperature spectra from lower temperature spectra clearly showed that the PPII content, indicated by the positive ellipticity around 220 nm, was increased with decreasing temperature in the polypeptides (fig. 2). On the other hand, the spectra of RP3-42H did not show such features (fig. 2) and were similar to those of RP3-42 with no marked secondary structure (Yamauchi et al. 1998). Therefore, the PPII-like structure observed for TDP24-2H and TDP25-4H was suggested to have emerged through the experimental evolution with selection based on binding affinity to DNA. In addition, these polypeptides are located on different main branches (fig. 1B). Hence, the emergence of the PPII-like structure was not branch-specific, suggesting that the structure appeared commonly, if not on all the evolved sequences.
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]) of the ancestral and evolved polypeptides. (A) RP3-42H at 4 °C (blue), 20 °C (green), and 60 °C (red). (B) TDP24-2H at 4 °C (blue), 20 °C (green), 60 °C (orange), and 70 °C (red). (C) TDP25-4H at 4 °C (blue), 20 °C (green), 60 °C (orange), and 70 °C (red). (D) YSLP1-1 at 5 °C (blue), 20 °C (green), 60 °C (orange), and 70 °C (red). (E) YSLP6-1 at 5 °C (blue), 20 °C (green), 60 °C (orange), and 70 °C (red). RP3-42H, TDP24-2H, and TDP25-4H were measured in HBST (pH 7.5) supplemented with 8% (v/v) NEB restriction enzyme diluent buffer A, and YSLP1-1 and YSLP6-1 reported previously (Yamauchi et al. 2002CD spectra of YSLP1-1 and YSLP6-1 were determined to examine the generality of the emergence of the PPII-like structure. YSLP1-1 and YSLP6-1 are polypeptides selected at the first and sixth generations, respectively, of experimental evolution starting from random polypeptides toward the highest affinity to a transition state analog for esterase reaction (Yamauchi et al. 2002
). These results showed that the spectra of YSLP6-1 have similar characteristics to those of TDP24-2H and TDP25-4H, but such characteristics were observed to only a small extent for YSLP1-1 (fig. 2), indicating an increase in the PPII-like structure during the experimental evolution. Together with the observations of the present study, these results support the theoretical proposition that structures coevolve gradually with function (Saito et al. 1997; Yomo et al. 1999), which is also a scenario proposed for protein diversification (James and Tawfik 2003), at the initial stages of protein evolution, and suggest that PPII is a common structure in primordial proteins. That is, our experimental evolution may have shown one of the common routes occurring in the early stages of protein evolution. Further development of structure and function of primordial proteins, which can be realized by continuing experimental evolution, will provide insights into the process of the development of protein evolution. |
Supplementary Material |
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Supplementary figures S1–S3 are available at Molecular biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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We thank Dr K. Ogasahara of the Institute for Protein Research, Osaka University, for technical guidance and discussions on CD spectroscopy; I. Nishina and W. Furutani of Center for Medical Research and Education, Graduate School of Medicine, Osaka University, for help and technical support with amino acid analysis and MALDI-TOF mass spectrometry; and also Dr T. Nakashima for valuable advice regarding phage display and in vitro selection experiments. This research was supported in part by "Special Coordination Funds for Promoting Science and Technology: Yuragi Project" and "Global COE (Centers of Excellence) Program" of the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
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Footnotes |
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Takashi Gojobori, Associate Editor
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