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 bee
Trang 1M I N I R E V I E W
Phage-display as a tool for quantifying protein stability
determinants
Joanne D Kotz1, Christopher J Bond2and Andrea G Cochran1
1
Department of Protein Engineering and2Medicinal 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 fromcombinatorial 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 ( 1010 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)
Trang 2accompanying 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 platformfor 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
froma 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 froma large pool of unfolded proteins In both cases
stable clones are identified fromthe 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 roomtemperature,
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 fromthe 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 fromthe 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.
Trang 3unfolding transitions For the mutant based on the third
round consensus sequence, the melting temperature (Tm)
was equal to that of wild type GB1 (81C) The Tmwas
somewhat reduced from wild type for the two dominant
fifth round clones (59C and 62 C; Table 1) In each case
the proteins were fully folded at roomtemperature 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 (VH) with
protein A was used as a surrogate for direct stability
measurements The VH domains in camelid heavy chain
antibodies are most similar to the classical VH3 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 60C, 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
L L R W F W R3 consensus 81
Trang 4export 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
randomturn 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 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 equilibriumconstant, 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 fromwhich 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)1relative 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
in CDR3, are shown in yellow The im age was generated using
SWISSPDBVIEWER
Trang 5The 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 fromthe Protein Data Bank
[25–27] These earlier studies indicated many statistically
significant deviations fromrandompairing, 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 forma nonhydrogen bonded pair, unlike residues 44
and 53 whose backbone amides are hydrogen bonded to one
another Nonhydrogen bonded pairs have different Caand
Cbdistances 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 fromDeGrado 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
2 kcalÆmol)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 froma larger pool of unfolded molecules [4] However, in distinguishing a more stable protein frommany 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 fromthose 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 fromthe 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 fromthe solvent exposed
face of the b-sheet are shown in gray.
Trang 6other 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 black box 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
equilibriumpopulation 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 shotgun phage-display to selections for
folding should expand the utility of combinatorial
muta-genesis and complement existing methodology [32] A major
advantage to the shotgun method is that it combines
strengths of traditional phage selections (e.g amplification
and discrimination of functional variants) with those of high
throughput screens (e.g adequate statistical sampling of smaller libraries) However, there are complications encoun-tered in studying folding by phage, namely the need for a selection that indirectly, yet cleanly, reports on stability Furthermore, as discussed above, there are certain param-eters (such as the stability of the starting host protein) that may require optimization in order to achieve good results It
is our belief that these concerns can be addressed and that phage-display will prove a useful, and fast, way to test quantitatively many hypotheses about protein structure
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