MINIREVIEW Phage-display as a tool for quantifying protein stability determinants Joanne D. Kotz 1 , Christopher J. Bond 2 and Andrea G. Cochran 1 1 Department of Protein Engineering and 2 Medicinal Chemistry, Genentech, Inc., South San Francisco, CA, USA To address questions of protein stability, researchers have increasingly turned to combinatorial approaches that permit the rapid analysis of libraries of protein variants. Phage- display has proved to be a powerful tool for analyzing protein stability due to the large library size and the robustness of the phage particle to a variety of denaturing conditions. With the B1 domain of protein G (GB1) and a camelid heavy chain antibody as model systems, we are using phage-display lib- raries to experimentally address questions that have generally been addressed in silico, either through computational stud- ies or statistical analysis of known protein structures. One effort has focused on identifying novel solutions to repacking the hydrophobic core of GB1, while maintaining stability comparable to the wild type protein. In a second study, a small set of substitutions in complimentarity-determining region 3 was found to stabilize the framework of the camelid antibody. Another major focus has been to obtain quanti- tative data on b-sheet stability determinants. We have suc- cessfully adapted a phage-display method for quantitating affinities of protein variants (shotgun alanine scanning) to analysis of GB1 stability. Using this method, we have ana- lyzed the energetic contributions of cross-strand side chain– side chain interactions. Finally, we discuss parameters to consider in using phage-display to discriminate subtle sta- bility differences among fully folded variants. Overall, this method provides a fast approach for quantitatively addres- sing biophysical questions. Keywords: beta sheet; hydrophobic core; phage-display; protein G; protein stability. Introduction Understanding determinants of protein stability is critical both for predicting the tertiary structure of a protein from an amino acid sequence, as well as for protein design. Rather than characterizing individual proteins with single mutations, or defined combinations of mutations, research- ers have increasingly been using selection and screening methods to investigate protein stability. In comparison to the labor-intensive process of generating and characterizing individual mutant proteins, these combinatorial approaches offer the important advantage of simultaneously generating libraries of protein variants, thus allowing a much larger number of mutations to be investigated. However, inter- preting the results from combinatorial experiments is not as straightforward as characterizing individual proteins. Con- sequently, results must be carefully assessed in light of the library design and selection pressure applied. Each screening or selection method, a number of which are discussed in this review series, will have inherent advantages and limitations that should be considered in addressing specific questions of protein structure. Phage-display is one selection technique that has been successfully applied to investigating protein stability [1,2]. In adapting phage-display from the more common selection for binding affinity, investigators have focused on mutating residues affecting protein stability, but not directly involved in ligand binding (Fig. 1). Proteins are selected that retain binding capacity, with the implicit assumption that a properly folded protein is required for an intact binding interface [3,4]. As a protein mutagenesis strategy, phage-display offers a number of important advantages. The technology for generating large libraries ( 10 10 members) has been well developed [5], permitting the simultaneous characterization of a relatively large number of mutants. In addition, the high in vitro stability of the phage particle [6] permits the use of a wide range of selection conditions. For example, investigators have used high temperature [7–9] and denat- urants [8,9] to increase selective pressure. Varying the stringency of selection conditions by these methods allows greater flexibility in experimental design and is particularly relevant to questions of protein stability. One limitation of the above approach is the requirement for a known binding partner with a binding interface that is unaffected by the mutations introduced. A number of researchers have developed strategies for circumventing this coupling of protein stability and function, relying on the greater susceptibility to proteolysis of unfolded proteins. An Correspondence to A. G. Cochran, Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080, USA. Fax: + 1 650 225 3734, Tel.: + 1 650 225 5943, E-mail: andrea@gene.com Abbreviations: CDR3, complimentarity-determining region 3; GB1, B1 domain of protein G; scFv, single chain variable fragment; V H , variable heavy chain. (Received 5 January 2004, revised 18 February 2004, accepted 5 March 2004) Eur. J. Biochem. 271, 1623–1629 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04076.x accompanying review by Bai & Feng will discuss the significant progress that has been made in developing these methods [10]. In this review, we will focus on studies in our laboratory investigating the effect of mutations on the stability of the B1 domain of Streptococcal protein G (GB1). One aspect of GB1 stability that we have addressed is the tolerance for mutations in the core of the protein. These studies provide a platform for comparing the results of combinatorial experimental studies with a system that has been well characterized computationally [11–14]. This work relies upon the binding of properly folded GB1 to the immuno- globulin Fc fragment to separate the few functional variants from a large number of unfolded proteins. A similar strategy is used by Bond et al. to characterize residues necessary for protein stability in single chain camelid antibodies [15]. By comparing the results of these studies using different proteins but a similar experimental design, we discuss the extent to which the most stable proteins are selected. Additionally, our laboratory has extended phage-based stability selections to include quantification of the relative contributions of amino acid substitutions to protein stabil- ity. Using the b-sheet of GB1 as a model system, we have asked whether a number of variants, all of which are folded under the conditions of the selection, can be recovered differentially based on varying stabilities. Remarkably, we have found that, at least in some circumstances, a quanti- tative correlation to biophysical data can be obtained from a statistical analysis of selected phage populations [16]. We also discuss experiments addressing the physical basis of this selection and the range of stabilities that can be differen- tiated. Selecting the most stable protein variants Two studies, one of GB1 and one of a camelid antibody, have employed phage-display to identify the most stable clones from a large pool of unfolded proteins. In both cases stable clones are identified from the selection and stability is confirmed when the individual proteins are characterized. However, it is not clear whether the globally most stable proteins encoded in the libraries are identified. The successes and limitations of this strategy, and ideas for increasing the selective pressure, are discussed below. Repacking the GB1 core We have begun to investigate by phage-display the tolerance to substitution in the hydrophobic core of GB1. One goal of this study was to compare phage-based strategies to computational methods. Based on the definitions of Mayo and coworkers, our library focused on core residues 5, 7 and 30, having less than 10% of the side-chain surface exposed to solvent [11], and boundary residues 16, 18 and 33, those which lie at the interface between the buried core and surface residues [12]. These residues are spatially close to one another and potentially in contact (Fig. 2). However, this library does not exactly duplicate the computational library because in the experimental system, residues near the Fc binding interface cannot be varied (for instance, core residues 3 and 39, and boundary residues 25 and 29 that were all changed in a hyperstable GB1 variant [12]). Based on the computational studies, core positions 5 and 30 were expected to be intolerant to substitution. In contrast, the core position 7 and boundary positions 16 and 18 have been shown to tolerate substitutions, with some variants even showing increased stability over the wild type protein [11,12,14]. Following three rounds of selection at room temperature, a consensus sequence began to emerge (Table 1). In agreement with previous work, the wild type residues were predominantly observed at positions 5 and 30, whereas alternative residues appeared to be tolerated, or even preferred, at all other library positions. For instance, arginine and tryptophan were frequently observed at positions 16 and 18, and tryptophan was preferred at position 33. Following two additional rounds of selection, a few particular sequences began to dominate the library. Three individual GB1 variants were expressed and purified for biophysical characterization; one of these differed only at boundary residues and was based on the consensus sequence from the third round of sorting, while two represented the most dominant clones from the fifth round of sorting. All three proteins underwent two-state thermal Fig. 2. Mutation of the GB1 core. Core residues (red) and boundary residues (blue) were randomized in one library. Other core residues [10] are shown in gray. This figure and Fig. 5 were generated from the NMR structure (PDB code 2GB1) [33] using INSIGHT II (Accelrys, San Diego, CA). Fig. 1. Phage-display as a method for selecting for protein stability. Folded proteins are retained, based on the formation of the three- dimensional structure necessary to form a functional binding interface. Unfolded proteins cannot bind and therefore are not selected. 1624 J. D. Kotz et al.(Eur. J. Biochem. 271) Ó FEBS 2004 unfolding transitions. For the mutant based on the third round consensus sequence, the melting temperature (T m ) was equal to that of wild type GB1 (81 °C). The T m was somewhat reduced from wild type for the two dominant fifth round clones (59 °Cand62°C; Table 1). In each case the proteins were fully folded at room temperature and retained IgG binding activity (J. D. Kotz & A. G. Cochran, unpublished results). To reduce the time required for computational analysis, only hydrophobic residues were allowed at core positions and only 16 residues were allowed at boundary positions (with cysteine, methionine, glycine and proline excluded in the in silico experiment) [11,12,14]. In contrast, in our experimental system all 20 amino acids were encoded at each position. Surprisingly, in the two clones that dominated the library, amino acids disallowed in the computational study were shown to result in stable, folded proteins. At the core position 7, serine was observed in one of the frequently observed clones. At boundary position 16, a proline occurred in the second clone investigated (Table 1). These results highlight the stability of the GB1 core to substitutions that may seem energetically unlikely, or even irrational, but that can be rapidly explored using a phage selection. Design of a heavy chain antibody scaffold A conceptually similar approach was employed by Bond et al. in the design of a camelid heavy chain antibody scaffold for use in constructing naı ¨ ve antibody libraries [15]. Here, the association of the variable heavy chain (V H )with protein A was used as a surrogate for direct stability measurements. The V H domains in camelid heavy chain antibodies are most similar to the classical V H 3 family and as such bind protein A with micromolar affinity. Further- more, the protein A binding site is distal to the former light chain interface and involves residues within the b-sheet structure (Fig. 3). As in the GB1-Fc system, the protein A– antibody interaction requires a correctly folded molecule, and therefore binding can be used as a direct readout for antibody stability. To adapt to the loss of the light chain, these heavy chain scaffolds rely on portions of complimentarity-determining region 3 (CDR3) to maintain structural integrity. This additional role of CDR3 complicated the design of heavy chain libraries for antigen binding selections by requiring a scaffold in which the structural residues of CDR3 were fixed. To identify these structurally important residues, potential heavy chain CDR3 scaffolds were evaluated by sorting a 17-residue CDR3 library against protein A. Following three rounds of sorting, 335 clones were isolated and sequenced. When purified proteins were individually characterized, the four most frequently observed clones had thermostabilities of approximately 60 °C, similar to those of other camelid heavy chain antibodies [15]. A crystal structure of one clone revealed that the residues selected at both ends of the CDR3 loop are ordered and interact with the former light chain interface, supporting the idea that these residues are structurally important (Fig. 4). Con- versely, the remainder of the loop is disordered, consistent with the observed tolerance to substitution at these loop positions (C. J. Bond, J. C. Marsters & S. S. Sidhu, unpublished results; [15]). To what extent are the most stable sequences selected? In both of the above studies, the dominant clones obtained after selection were shown to be stable and well-folded proteins. However, in the GB1 study we failed to identify new variants with significantly increased stability compared to the starting protein. Furthermore, the dominant round five clones were not as stable as the wild type sequence and therefore were not the most stable proteins encoded in the library. This observation high- lights an important caveat when using phage-display. The sequences selected at each round are influenced by a variety of factors including codon usage, expression levels, Fig. 3. Structure of the Protein A–Fab complex (PDB code 1DEE) [34]. The heavy chain is colored blue and the light chain green. Protein A (orange) interacts with the heavy chain on the side opposite to the light chain interface and is represented using the program SWISSPDBVIEWER . Table 1. Experimental repacking of the GB1 core. A representative sample of clones obtained after three rounds of selection for GB1 binding is shown at top. Proteins that were individually purified and characterized are shown in bold. Position Round of sorting %of clones T m (°C) 5 7 16 18 30 33 LLRRFW3 1 LLR WF W3 1 LFRWFF3 1 LI R WL W3 1 LSTLFW3 1 LLTWFF3 1 L L R W F W R3 consensus 81 LSI K F L 5 30 59 LYP V F M5 5 62 LLTTFYWT 81 Ó FEBS 2004 Phage-display for quantifying protein stability (Eur. J. Biochem. 271) 1625 export to the surface of the phage and affinity for the target protein. Thus, although a folded protein is a minimum requirement, phage-display selection may not depend solely on protein stability. To address this limitation, the selection can be made more dependent on protein stability by destabilizing the host. For example, to test the range of turn sequences permitted in stable proteins [7], DeGrado and coworkers generated random turn sequences in GB1 host proteins of different stabilities; these libraries were screened for IgG competent binders at either room temperature or elevated temperature. They observed no sequence preference for turns in the wild type host, whereas clear sequence preferences were observed in destabilized hosts. As the stringency of the screen increased, the functional solutions increasingly resembled the wild type sequence and turns that are most commonly found in proteins. Highlighting the effectiveness of the screen, they confirmed biochemically that IgG binders obtained under the most stringent conditions were signifi- cantly more stable than nonbinders [7]. In a different approach, Plu ¨ ckthun and coworkers directly compared the use of temperature or denaturant as a selective pressure to improve the stability of single chain variable fragment (scFv) by phage-display [8]. In their study, the phage- displaying scFv variants were subjected to high temperature, denaturant or no stress prior to selection. High-temperature stress resulted in the most sequence convergence and the most stable clone analyzed. The authors concluded from this observation that, at least in their system, temperature stress was much more stringent than incubation with denaturant. However, this may not be general; instead, it may be a consequence of the irreversibility of thermal denaturation and the reversibility of chemical denaturation for scFV [8]. In the case of GB1, with known hyperstable variants, we intend to compare the results of increasing the selective pressure through each of these methods. Hopefully, as additional systems are investigated, a general under- standing of the pressure needed for a given stringency of selection will emerge. Quantifying protein stability A major focus of our laboratory has been extending the use of phage-display to allow ranking of the stabilities of individual proteins in a pool of folded variants. In addition, a rapid method for quantitatively, and simultaneously, characterizing a large number of mutants would greatly aid in understanding the effects of complex interactions on stability, for instance, the effect of long range tertiary contacts on b-sheet formation. We discuss recent advances in the use of phage-display to probe the energetics of b-sheet formation, as well as progress in understanding important experimental variables of the method. Analyzing stability determinants in the b-sheet of GB1 The potential for obtaining quantitative biophysical infor- mation from phage-display was suggested by a new method called alanine shotgun scanning, which analyzes the ener- getic contribution of residues at a binding interface. Sidhu, Weiss and coworkers [18–20] have treated the observed frequency ratios of residues at a given position (i.e. wild type/alanine ratio) as an equilibrium constant, which is then used to calculate the relative free energies of binding for different protein variants. Relative energies calculated from this distribution data have been shown to correspond directly with data obtained for individual purified mutants [18]. Thus, shotgun scanning provides a rapid method for using phage-display to quantify changes in affinity. In order to apply this method to the ranking of protein stabilities, the well-established b-sheet model system GB1 seemed an ideal initial target [13]. A library was constructed varying two cross-strand residues, 44 and 53 (Fig. 5). These positions were guest sites in published mutagenesis studies from which b-sheet propensity scales have been developed [21–24]. In both protein mutagenesis and phage studies, the host protein included the I6A mutation, resulting in the destabilization of the protein by  2.5 kcalÆmol )1 relative to wild type GB1. Following a binding selection at room temperature, individual clones were sequenced. From the observed residue distributions at position 53, a phage-based stability scale was calculated. Strikingly, this scale correlated quantitatively with the published propensity scale derived from thermal stability measurements [16]. Fig. 4. Structure of the evolved heavy chain scaffold [17]. The frame- work regions are colored grey while CDR3 is colored red. Residues critical to scaffold stablility, both at the former light chain interface and inCDR3,areshowninyellow.Theimagewasgeneratedusing SWISSPDBVIEWER . 1626 J. D. Kotz et al.(Eur. J. Biochem. 271) Ó FEBS 2004 The use of an unusually trivial library, in which just two surface positions were varied to all amino acids, permitted the rapid analysis of the energetic contribution of side chain–side chain interactions at a single surface site. This analysis could be compared to earlier studies of residue pairing in diverse b-sheets from the Protein Data Bank [25–27]. These earlier studies indicated many statistically significant deviations from random pairing, the nature of which depended to some extent on the exact method of data normalization. In contrast, the data obtained by the phage- display method (that do not require normalization) sugges- ted only minor energetic contributions from most side chain interactions [16]. This rapid analysis of many amino acid pairs demonstrates the power of using combinatorial approaches to address questions of protein stability, where a large number of interactions must be characterized to understand the underlying trends. Current work in our laboratory is directed toward extending the studies of b-propensities and side chain–side chain interactions in b-sheets. We are now creating a library at positions 6 and 15 of the GB1 b-sheet. These two side chains form a nonhydrogen bonded pair, unlike residues 44 and 53 whose backbone amides are hydrogen bonded to one another. Nonhydrogen bonded pairs have different C a and C b distances than hydrogen bonded pairs and therefore may have different residue preferences and potential for side chain–side chain interactions. Initial results suggest that the selected residue distributions correlate well with a conven- tional stability scale for position 6, and we are in the process of analyzing the relationship between the two positions (J. D. Kotz & A. G. Cochran, unpublished results). Importantly, these results suggest that the phage-display method will be generally applicable to quantitative compar- isons of protein stabilities. Importance of host stability for selection The investigation of turn stability from DeGrado and coworkers (discussed above) emphasized the importance of host stability in tuning the stringency of a selection or screen [7]. In our effort to rank protein variants of very similar stabilities, we will probably need to achieve a balance between a very stable host, whose folded population would be predicted to be insensitive to stability changes, and a very unstable host that would not allow characterization of destabilizing mutations. In our current studies of positions 6 and 15 of the GB1 b-sheet, we have used hosts of two different stabilities (wild type and a mutant destabilized by  2kcalÆmol )1 ). By comparing results from the two host proteins, we intend to characterize the host stability range necessary for obtaining quantitative data. Surprisingly, we have found that variants that should be fully folded at the temperature of selection can nevertheless be discriminated, raising questions about the basis for selection [16]. Physical basis for ordering stabilities As described above, a binding selection relies on the requirement that a protein be folded in order to functionally interact with a binding partner. This is a powerful method for the isolation of rare folded variants from a larger pool of unfolded molecules [4]. However, in distinguishing a more stable protein from many other stable proteins, the physical basis for the selection is not as clear. The most straightfor- ward idea is that each protein variant is in equilibrium between the folded and unfolded state with only the folded state being competent for binding [2]. That is, folding is thermodynamically coupled to binding, resulting in an apparent affinity change as the fraction of folded molecules changes [28]. A second possibility is that many proteins in the pool are fully folded but that enhanced protein stability leads somehow to higher target affinity, increasing the likelihood of recovery in the binding selection. Finally, more stable proteins may be displayed at a higher level on the surface of the phage, but then once displayed, retained with equal probability during the interaction with the binding partner. To distinguish changes in display level from selections requiring interaction with the specific binding partner (stability- or affinity-based), one could divide a phage library in half and sort one half against a binding partner and the other half against an expression tag. A comparison of the sequences obtained from these two different selections should reveal any display bias. Alter- natively, a Western blot could be used to directly probe the display levels of selected protein variants. To distinguish affinity-based selections from those based solely on protein stability, the affinities of purified mutant proteins for target can be measured by standard methods (immunoassay or surface plasmon resonance) at the temperature of the phage selection (or at temperatures at which the variants are fully folded); in studies with GB1, it does not appear that sufficient affinity differences exist among the folded variants to explain their differential selection (J. D. Kotz & A. G. Cochran, unpublished results) [16,24]. Analyzing the less stable GB1 variants Another potential limitation in quantifying stability by phage-display results from the inherently larger number of sequences observed for the more stable clones. For example, in the analysis of side chain interactions at positions 44 and 53 in GB1, the hydrophobic amino acids are more stabilizing, and thus they occur much more frequently than Fig. 5. Quantifying b-sheet stability. The hydrogen-bonded pair (red) and the nonhydrogen-bonded pair (blue) were varied in separate phage-display libraries. Surrounding residues from the solvent exposed face of the b-sheet are shown in gray. Ó FEBS 2004 Phage-display for quantifying protein stability (Eur. J. Biochem. 271) 1627 other amino acids. Therefore, even with  1200 sequences, many of the 400 possible pairwise combinations are not expected to occur very frequently (if at all). As a result, even though charged side chain–side chain interactions are thought to be energetically significant [29,30] (and we did observe a number of oppositely charged pairs), charged amino acids did not occur frequently enough in our GB1 data set for observed pair correlations to be statistically significant. One could imagine addressing this issue by sequencing 10- to 100-fold more clones. However, it should be possible instead to increase representation of these amino acids by tailoring the initial library (by a different choice of degenerate codon), thereby eliminating the dominant, more stabilizing residues. Conclusions and future prospects It has long been appreciated that phage libraries are a rich source of unexpected functional diversity. Because of the very large library sizes that can be achieved, it is tempting to maximize the number of positions varied and then carry out many rounds of selection, in order to increase the chance of identifying a rare, highly functional clone. Although it is often possible to identify exciting new molecules, this approach introduces a Ôblack boxÕ aspect to the use of phage technology. In contrast, large scale screens can be very useful when asking certain quantitative questions, for example, what fraction of library members exhibits a property of interest? Unfortunately, such screens generally provide only crude measures of the degree to which a variant exhibits the property. Thus, it is difficult to use screens to answer many questions about proteins that are traditionally addressed by conventional site-directed muta- genesis and assays of purified variants. This is particularly relevant to the study of protein stability, in which detailed thermodynamic comparisons are often required. One solu- tion is to design screens that yield a reliable, quantifiable output parameter that reports on the stability of library members [31]. We have chosen instead to modify our use of phage selection technology. The discovery that phage-display can be used quantita- tively to report on affinity is an exciting new development [18,20]. It appears that once nonfunctional background phage are eliminated from a library through a few rounds of selection, the remaining phage essentially represent an equilibrium population of more and less functional vari- ants, allowing the use of statistical thermodynamics to rank free energy differences. Because DNA sequencing is now relatively routine, it is straightforward to sample the selected phage population sufficiently to identify residues important for binding and to provide a reliable estimate of how important they are. For binary mutagenesis (e.g. wild type vs. alanine), only a few hundred sequences are needed to characterize most interfaces [18], and an experiment of this type can be conducted inexpensively in a couple of weeks. The extension of ÔshotgunÕ phage-display to selections for folding should expand the utility of combinatorial muta- genesis and complement existing methodology [32]. 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MINIREVIEW Phage-display as a tool for quantifying protein stability determinants Joanne D. Kotz 1 , Christopher J. Bond 2 and Andrea G. Cochran 1 1 Department. b-sheet stability determinants. We have suc- cessfully adapted a phage-display method for quantitating a nities of protein variants (shotgun alanine scanning)