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Department of Chemistry, Princeton University, Princeton, New Jersey 08544-1009, USA
Reprint requests to: Michael H. Hecht, Department of Chemistry, Princeton University, Princeton, NJ 08544-1009, USA; e-mail: hecht{at} princeton.edu; fax: (609) 258-6746.
(RECEIVED August 9, 2002; FINAL REVISION October 7, 2002; ACCEPTED October 9, 2002)
1 These authors contributed equally to this work. 
2 Present address: R.W. Johnson Pharmaceutical Research Institute, Route 202, P.O. Box 300, Raritan, NJ 08869, USA.
3 Present address: Department of Chemistry, Brown University, Providence, RI 02912, USA.
Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.0228003.
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Abstract |
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-helical and quite stable; (2) four of the five contain an abundance of tertiary interactions indicative of well-ordered structures; and (3) one protein forms a well-folded structure with native-like features. The proteins from this new 102-residue library are substantially more stable and dramatically more native-like than those from an earlier binary patterned library of 74-residue sequences. These findings demonstrate that chain length is a crucial determinant of structural order in libraries of de novo four-helix bundles. Moreover, these results show that the binary code strategy—if applied to an appropriately designed structural scaffold—can generate large collections of stably folded and/or native-like proteins. Keywords: Protein design; binary patterning; de novo proteins; native-like protein structure; combinatorial library; four-helix bundle
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Introduction |
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Initial application of the binary code strategy focused on designing a library of 74-residue sequences targeted to fold into four-helix bundles (Kamtekar et al. 1993). Purification and characterization of >50 proteins from the initial collection demonstrated that virtually all sequences indeed folded into -helical structures (Kamtekar et al. 1993; Roy and Hecht 2000).
Because of its combinatorial nature, the binary code strategy does not allow explicit design of specific interresidue interactions. Because unique packing cannot be designed a priori, it is reasonable to question whether "native-like" structures can nonetheless be isolated from binary code libraries a posteriori. Searches for well-folded proteins in the original 74-residue library showed that several proteins possessed some native-like characteristics (Roy et al. 1997a, 1997b; Rosenbaum et al. 1999; Roy and Hecht 2000). However, most proteins in the initial collection formed fluctuating structures, reminiscent of molten globule intermediates.
Why did most sequences from the original collection fail to form native-like structures? One might postulate that fluctuating "molten" structures are exactly what should be expected from a combinatorial strategy that precludes explicit design of specific sequences with predetermined side-chain interactions. However, the alternative result—native-like structures—might have been predicted by numerous studies demonstrating that a well-folded structure can be specified by many different amino acid sequences (Dill 1985; Chothia and Lesk 1987; Bowie et al. 1990; Matthews 1993; Bromberg and Dill 1994; Axe et al. 1996; Gassner et al. 1996; Munson et al. 1996; Riddle et al. 1997). Comparisons of evolutionarily related sequences, theoretical studies using simplified models, and extensive mutagenesis experiments have led to the realization that protein structures are robust, and explicit design of "jigsaw puzzle" packing may not be necessary. For example, Matthews and coworkers replaced up to 10 residues in the core of T4 lysozyme with methionines and found—in contrast to the predictions of the jigsaw puzzle model—that the multiply substituted proteins were active and cooperatively folded (Gassner et al. 1996).
These and other findings (Dill 1985; Chothia and Lesk 1987; Bowie et al. 1990; Lau and Dill 1990; Behe et al. 1991; Matthews 1993; Bromberg and Dill 1994; Axe et al. 1996; Gassner et al. 1996; Munson et al. 1996; Riddle et al. 1997) led us to question whether the tendency of the original binary code proteins to form fluctional structures might not be a failure of the binary code strategy per se but rather a shortcoming of the designed structural scaffold used in its initial implementation. In particular, we questioned whether the -helices specified by our original scaffold might simply be too short. We reasoned that in the context of the binary code strategy, which cannot specify side chain packing a priori, it might be advantageous to use a scaffold that encodes longer -helices, and hence, larger interhelical interfaces. Here, we describe the design of a second-generation scaffold, in which the four -helices are 50% longer. Characterization of several proteins arbitrarily chosen from the new library demonstrates that they are all significantly more stable, and most of them (four out of five) are substantially more native-like than proteins from the original binary code library.
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Results |
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-helices typically longer than 20 residues (Y. Wei and M.H. Hecht, unpubl.). To assess whether a longer structural scaffold would favor well-folded structures over molten globules, we constructed a second-generation library of 102-residue sequences. This new library was not constructed "from scratch." Instead, to stringently test whether the redesigned features are sufficient to convert a fluctional protein into a well-ordered structure, we chose a typical molten globule-like protein (sequence #86) from the original 74-residue library (Kamtekar et al. 1993) as the starting point for designing the second-generation elongated library. The new library of sequences was constructed in two steps. In the first step, two minor modifications were incorporated: (1) a tyrosine was inserted after the initiator methionine to facilitate concentration determinations and also to prevent methionine removal in vivo (Bachmair et al. 1986), and (2) a glycine dipeptide was inserted into the central interhelical turn in place of a proline. Proline was undesirable because cis/trans isomerism could lead to multiple (rather than unique) conformations. The modified sequence with these two changes (called n86) served as the template for constructing the second-generation binary code library.
In the second step, sequence n86 was elongated by inserting combinatorially diverse sequences between -helices 1 and 2, and between -helices 3 and 4. Each of the four -helices was elongated from 14 residues to 20 residues (not including N- and C-caps) in accordance with the binary code patterning. The overall design of the structural scaffold for the second-generation library is summarized in Figure 1
. Construction of the second-generation library of synthetic genes using combinatorial mixes of DNA codons encoding polar, nonpolar, N-cap, C-cap, and turn residues is described in Materials and Methods.
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. The proteins were purified using published methods (Kamtekar et al. 1993; Johnson and Hecht 1994; Roy and Hecht 2000), and their identities were confirmed by electrospray mass spectrometry (Table 1).
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-helical, and stable
), even at the relatively high concentrations used for NMR spectroscopy. The proteins all formed -helical structures, with circular dichroism (CD) spectra showing the typical -helical signatures including a maximum at 190 nm, and minima at 208 and 222 nm (not shown). The magnitudes of the minima at 222 nm (Table 1) are as expected for four-helix bundles.
The stabilities of the second-generation proteins are compared to the parental protein, n86, in Figure 3. All five of the newly designed 102-residue proteins are substantially more stable than the parental n86 protein. The free energies stabilizing the folded state relative to the unfolded state are approximately two- to threefold more favorable for the elongated proteins relative to the parental n86 protein (Table 1).
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Figure 4 compares the NOESY spectrum of the parental n86 protein with the spectra of the five second-generation proteins. The spectrum of protein n86 shows poor chemical shift dispersion, only a few distinguishable interresidue NH-to-NH contacts, and only a limited number of side-chain interactions. Thus, protein n86 does not form a well-folded structure. The spectrum of one second-generation protein, S-23, although better than that of n86, also does not display the dispersion, resolution, or abundance of NOE cross-peaks that would be expected for a well-folded structure.
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15N,1H-HSQC NMR spectroscopy
Additional information about the native-like versus molten globule-like properties of a protein can be obtained from heteronuclear NMR spectroscopy. Figure 5 compares the 15N,1H-HSQC spectra of the second-generation proteins with the spectrum of the parental n86 protein. As expected for proteins with different sequences, the HSQC spectra of the five second-generation proteins range in quality. Consistent with the homonuclear results described above, the HSQC spectrum of protein S-23 is only slightly better than that of the parental protein, n86. However, the other four second-generation proteins (S-213, S-285, S-824, and S-836) yield 15N,1H-HSQ spectra that are substantially better than n86: All four display peak dispersion (in both dimensions) that is superior to n86, and is comparable to the dispersion seen for many natural -helical proteins. Moreover, in contrast to n86, which contains only a few well-resolved cross-peaks, the spectra of these four second-generation proteins contain numerous well-resolved cross-peaks. Based on these spectra, it is clear that the structures of these arbitrarily selected second-generation proteins are substantially more ordered than that of the parental protein, n86.
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13C,1H-HSQC NMR spectroscopy
Although the 15N,1H-HSQC spectra described above (and shown in Fig. 5) suggest that the second-generation proteins form ordered structures, these spectra report primarily on the environment of the backbone. To distinguish well-folded structures from those with extensive local mobility, it is also necessary to analyze the environments of the side chains—especially those in the hydrophobic core. As described recently by Walsh et al. (2001a, b), the degree of side-chain order can be assessed by natural abundance 13C,1H-HSQC NMR spectroscopy.
We measured natural abundance 13C,1H-HSQC NMR spectra for the five second-generation proteins. The methyl 13C,1H correlations for these proteins are shown in Figure 6. In these spectra, the 
and 
methyl resonances of isoleucine side chains appear in the 13C dimension (F1 in Fig. 6) at 
11 and 15 ppm, respectively. Methyl groups from the side chains of Val, Met, Leu, and Thr occur between 18 and 24 ppm.
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) showed poor dispersion and few NOEs. However, the 13C,1H-HSQC spectra of the four other second-generation proteins display well-resolved peaks and good dispersion. Although we have not yet assigned the peaks in these spectra, it is clear from the isoleucine regions, that all (or almost all) of the isoleucine methyl resonances are well resolved. For example, for protein S-824, there are five isoleuecines in the sequence (see Fig. 2), and five resonances can be seen in both the methyl and the methyl regions. More detailed studies of the structures and dynamics of these proteins are in progress.
Differential scanning calorimetry
The NMR experiments described above were performed to assess the structural properties of the de novo proteins. An orthogonal method for distinguishing between molten globules and native-like structures is differential scanning calorimetry (DSC), which measures their thermodynamic properties. The thermal denaturation of molten globules typically occurs over a broad temperature range with a relatively low 
H. In contrast, native structures denature cooperatively with relatively sharp transitions and larger enthalpies.
Figure 7 compares the thermal denaturation of protein n86 with the second-generation protein S-824. The elongated protein is significantly more thermally stable. Moreover, it denatures over a relatively narrow temperature range, and with a larger H. Because of the high thermal stability of S-824, the denaturation was not complete even at 110°C. Therefore, calculation of a precise H was not possible. Nonetheless, by assuming the peak is symmetric, we estimate a H between 110 and 130 kcal/mole.
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H. Therefore, when interpreting our thermodynamic results, it is important to compare the measured H for protein S-824 to the native and molten globule states of a reference protein of similar size and structure. Cytochrome b562 is a natural four-helix bundle containing 106 residues (compared to 102 in protein S-824). The H reported for the denaturation of native cytochrome b562 is 104 kcal/mole (Robinson et al. 1998). This is similar—or slightly lower—than the 120 kcal/mole we estimate for S-824. The H of S-824, however, is two- to threefold greater than the H (46 kcal/mole) reported for the denaturation of the molten globule form of apocytochrome b562 (Feng et al. 1994).
The enthalpy of denaturation for protein S-824 is also approximatly threefold greater than that measured for the parental sequence n86. This difference is considerably larger than would be expected solely from the difference in size (102 residues versus 75 residues, that is, 33%). Thus, the calorimetrically measured thermodynamic properties of S-824 (Fig. 7) are consistent with the spectroscopically measured structural properties (Figs. 4–6) in demonstrating that this second-generation protein is significantly more stable and more native-like than the parental protein, n86.
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Discussion |
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) and nonpolar (•) amino acids demonstrated that designs based on the -helical periodicity •••••• specify proteins that are soluble and -helical (Kamtekar et al. 1993; Roy et al. 1997a, 1997b; Rosenbaum et al. 1999; Roy and Hecht 2000). Among these -helical collections, de novo proteins that bind cofactors and catalyze reactions occur quite frequently (Moffet et al. 2000, 2001; Moffet and Hecht 2001). More recent work showed that libraries based on the ß-strand pattern ••• yield proteins that form ß-sheet structures. By varying either the details of the ß-sheet design, or the conditions of the experiment (or both) we have produced collections of binary patterned ß-sheet proteins that form either monomeric structures, amyloid-like fibrils, self-assembled monolayers, or template assembled biomaterials (West et al. 1999; Xu et al. 2001; Brown et al. 2002; Wang and Hecht 2002). Although these earlier studies demonstrated that binary patterning can be used to guide the design of various protein structures and functions, a nagging question remained: can the binary code strategy also produce libraries of sequences that recapitulate the structural and thermodynamic properties of well-folded native proteins?
In assessing the potential of the binary code strategy to produce well-folded structures, we considered two opposing hypotheses: (1) Well-ordered structures may be difficult to achieve and therefore must either be designed explicitly or selected evolutionarily. If this hypothesis were correct, then binary patterned libraries would rarely yield well-folded proteins. (2) Alternatively, for a given structural scaffold, well-ordered structures may be achievable with many different combinations of side chains. This hypothesis is supported by statistical studies demonstrating that in natural proteins apolar side chains have little or no inherent preference for specific packing interactions, and "can pack together efficiently in a large number of ways" (Behe et al. 1991). This would lead to the expectation that well-folded structures would occur relatively frequently in binary patterned libraries.
Which of these two hypotheses is actually correct depends, at least in part, on the structural scaffold used in the design. Some scaffolds will rarely, if ever, yield well-folded structures. Because our earlier -helical library (Kamtekar et al. 1993) was based on a scaffold considerably shorter than found in natural four-helix bundles (74 versus >100 residues), it became apparent that a realistic test of the binary code strategy required the design of a second-generation library using a longer structural scaffold.
The current work describes the construction of this second-generation library. Five proteins from the new library were purified and characterized. The structural and thermodynamic methods used to analyze these proteins demonstrate that (1) all five are -helical and quite stable; (2) four of the five adopt structures that are reasonably well ordered; and (3) at least one of these proteins, S-824, is very well ordered and appears to fold into a structure that is native-like or near native-like.
The designation of a protein structure as "native-like" or "near native-like" is not entirely black and white. In the early days of protein design (Regan and DeGrado 1988; Hecht et al. 1990), a structure capable of producing the data shown in Figures 3–7 would have been considered a successful native-like de novo protein. However, during the past decade, as protein designers have gained experience, and as methods for probing structural rigidity have become more sophisticated, the goal of producing native-like de novo proteins has become somewhat of a moving target. For example, DeGrado and coworkers (1999) reported the NMR structures of a three-helix bundle, which according to several criteria appeared native-like (Walsh et al. 1999). However, more recent analyses of the response of this protein to mutations, as well as NMR studies of side chain dynamics, has demonstrated that the protein is both more malleable and more dynamic than those natural proteins that have been examined at the same level of detail (Walsh et al. 2001a, 2001b). Thus, a detailed understanding of the native-like properties of the second-generation binary code proteins will require further studies of their structures and dynamics. Such studies are underway.
Despite our incomplete knowledge of the detailed structures and dynamics of these proteins, it is clear from the data presented above that proteins from the new library are substantially more stable and dramatically more ordered than the parental proteins from which they were derived. The five proteins characterized in this study were chosen arbitrarily: Neither genetic selections in vivo nor high throughput screens in vitro were required to select these sequences from the overall library. Hence, they presumably represent a fairly unbiased sampling of the second-generation library. Therefore, our observation that well-ordered structures occur in such a small sampling suggests that the first of the two hypotheses enumerated above is false: Structures that are reasonably well ordered are not difficult to achieve; they need not be selected evolutionarily or designed residue by residue.
Although four of the five second-generation proteins appear well ordered, one protein (S-23) is not. Because the identities of individual amino acids clearly play a role in side chain packing, some combinations of amino acids will not be compatible with well-folded structures. Therefore, even for a well-designed scaffold, some binary patterned sequences will not fold into well-ordered structures: in the current library, sequence S-23 is closer to a molten globule.
Although S-23 is the least native-like of the five second-generation proteins (Figs. 4–6), it has the highest midpoint for guanidine denaturation (Fig. 3). Thus, as noted previously (Betz et al. 1993), enhanced stability alone does not prove a structure is more ordered or more native-like.
Although the reason that S-23 is an outlier is not yet known, we consider two possibilities: (1) S-23 is the only sequence with no tryptophans. Trp was shown recently to induce native-like structure in otherwise flexible chains (Klein-Seetharaman et al. 2002), and the absence of Trp in S-23 may tip the balance towards the molten globule state. (2) S-23 contains acidic side chains at positions 27, 28, 79, and 80. It is the only sequence with negative charges at all four of these sites. These positions are designed to be in the interhelical turns at the "top" of the four-helix bundle (Fig. 1). They would be in close proximity in the designed structure, and the local concentration of uncompensated negative charges may disfavor an ordered structure.
Despite the occasional molten globule, our results indicate that the second of the two opposing hypotheses enumerated above is closer to the truth: for a given structural scaffold, well-folded structures can be specified by many different amino acid sequences. Consequently, when binary patterning is applied to an appropriately designed structural scaffold, the resulting libraries contain a relatively large number of well-ordered and/or native-like structures.
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Materials and methods |
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Sequence n86 was then used as the template for constructing a new combinatorial library of 102-residue sequences. The newly inserted regions of sequence are shaded gray in Figure 1. The inserted regions were designed as follows:
The new regions of sequence (gray in Fig. 1) were incorporated using two sequential steps of full plasmid PCR to insert long stretches of combinatorially diverse sequences first between -helices 1 and 2, and then between -helices 3 and 4. The steps involved in this construction are shown schematically in Figure 8.
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Size-exclusion chromatography
Elution times were measured using a Superdex 75 HR/10/30 gel filtration column (Pharmacia). Size-exclusion chromatography was performed at the same concentrations as NMR experiments in 50 mM sodium acetate-acetic acid buffer, pH 4.0.
Circular dichroism spectroscopy
CD measurements were performed at 20°C in 10 mM NaPO4 (pH 6.8), 40 mM NaF using an Aviv model 62 DS spectrapolarimeter.
NMR spectroscopy
Spectra were acquired at 25°C and 600 MHz on a Varian Unity/INOVA spectrometer, using WATERGATE solvent suppression (Piotto et al. 1992). Data processing was done using NMRPipe (Delaglio et al. 1995), while NMRView (Johnson and Blevins 1994) was used for visualization and data analysis. Protein concentration was ca. 1 mM in a buffer containing 50 mM sodium acetate/acetic acid at pH 4.0. (Several experiments run at pH = 6.8 yielded similar results.)
NOESY Spectra were acquired in 95%H2O/5%D2O with a 150-msec mixing time using gradients for suppression of radiation damping during t1 and for artifact suppression (Sklenar 1995). Data acquisition parameters for the NOESY spectra were as follows: The spectral window was 8 kHz with the carrier positioned at the water resonance (4.769 ppm). 4K* and 480* data points were collected in t2 and t1, respectively (t2[max] = 511 ms, t1[max] = 60 msec) with 16 scans collected for each FID. Data processing included digital filtering for residual solvent signal suppression, apodization with combined shifted cosine and Gaussian functions, zero filling in both dimensions, reconstruction of two missing complex data points in t1 by backwards linear prediction (LP), and automated polynomial baseline correction in both dimensions. The final size of the frequency domain data was 8K x 2K data points.
1H,15N HSQC spectra were recorded on uniformly 15N-labeled proteins, prepared from cultures grown in minimal medium with 15N-labeled ammonium chloride as the sole nitrogen source. HSQC spectra were recorded using sensitivity enhanced gradient selected HSQC technique (Kay et al. 1992). The spectra were acquired with an 8 kHz spectral window for 1H, positioning the carrier on the water resonance (4.769 ppm), and 2 kHz frequency range for 15N (centered at 118.56 ppm). 1024* and 128* data points were collected in t2 and t1, respectively (t2[max] = 128 msec, t1[max] = 64 msec), averaging eight transients for each FID. Data processing included digital filtering for suppression of the residual solvent signal, combined shifted cosine and Gaussian apodization, and zero filling in both dimensions. Final data size was 2K x 2K data points.
Natural abundance 13C,1H-HSQC spectra were run at 599.5 MHz (1H) and 150.8 MHz (13C) using the pulse sequence in ProteinPack provided with the Vnmr software (Varian, Inc.). The carrier was positioned at 4.769 ppm (water resonance) for 1H and 35.00 ppm for 13C. Spectral windows were 8 KHz for 1H and 12 kHz for 13C. In t2 1024* points were collected (t2[max] = 126 msec) averaging 64 scans for each FID, while 256* data points were acquired in t1 (t1[max] = 21 msec). Data processing included digital filtering for removal of the residual solvent signal, combined shifted cosine and Gaussian apodization, and zero filling prior to Fourier transform. Final data size was 2K x 2K data points.
Differential scanning calorimetry
Scans were performed on a MicroCal. MC-2 calorimeter. Samples were in 50 mM NaAc buffer, pH 4.1. Calorimetric analysis of protein S-836 (not shown) demonstrated that it denatures at even higher temperatures than protein S-824. Proteins S-23, S-213, and S-285 denature at higher guanidine concentrations than either S-824 or S-836, and no attempts were made to measure their calorimetric denaturations.
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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