Philip Evans - Academia.edu (original) (raw)

Papers by Philip Evans

Folding and Design, 1996

Kinetic studies of folding sometimes reveal very rapid spectroscopic changes that may indicate th... more Kinetic studies of folding sometimes reveal very rapid spectroscopic changes that may indicate the population of intermediates, but it is difficult to elucidate in detail the nature of the interactions involved. In this review, we focus on one important aspect of this problem: how to probe the nature and extent of clustering of hydrophobic sidechains. As the information obtainable from different experimental approaches is outlined, it becomes clear that a combination of methods is likely to be necessary to build up a reasonable picture of early folding events.

Trends in Biochemical Sciences, 1994

Hen lysozyme is one of the best characterized and most studied of all proteins. Recently, we have... more Hen lysozyme is one of the best characterized and most studied of all proteins. Recently, we have used a range of different methods to examine the events involved in the in vitro folding pathway of this protein. In this review we show that, by combining complementary techniques, it has been possible to piece together a detailed model for the folding of this enzyme. Important questions prompted by this work are highlighted and we then propose some ideas consistent with our data, as well as those of others, which we believe begin to provide insight into one of the most intriguing of structural problems in biology--how proteins can achieve their complex native forms from disordered denatured states.

Trends in Biochemical Sciences, 1993

Proteins: Structure, Function, and Bioinformatics, 1992

The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme... more The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30°C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 105–107 times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35°C, by lowering the stability of the native state through selective cleavage of the Cys‐6–Cys‐127 disulfide crosslink (CM6−127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation...

Nature, 1992

Analysis of the folding of hen lysozyme shows that the protein does not become organized in a sin... more Analysis of the folding of hen lysozyme shows that the protein does not become organized in a single cooperative event but that different parts of the structure become stabilized with very different kinetics. In particular, in most molecules the alpha-helical domain folds faster than the beta-sheet domain. Furthermore, different populations of molecules fold by kinetically distinct pathways. Thus, folding is not a simple sequential assembly process but involves parallel alternative pathways, some of which may involve substantial reorganization steps.

Journal of Molecular Biology, 1998

The folding and unfolding kinetics of the N-terminal domain of the ribosomal protein L9 have been... more The folding and unfolding kinetics of the N-terminal domain of the ribosomal protein L9 have been measured at temperatures between 7 and 85 C and between 0 and 6 M guanidine deuterium chloride. Stopped-ow¯uorescence was used to measure rates below 55 C and NMR lineshape analysis was used above 55 C. The amplitudes and rate pro®les of the stopped-¯ow¯uorescence experiments are consistent with a two-state folding mechanism, and plots of ln(k) versus guanidine deuterium chloride concentration show the classic v-shape indicative of two-state folding. There is no roll over in the plots when the experiments are repeated in the presence of 400 mM sodium sulfate. Temperature and denaturant effects were ®t simultaneously to the simple model k D exp(ÀÁG { /RT) where ÁG { represents the change in apparent free energy between the transition state and the folded or unfolded state and D represents the maximum possible folding speed. ÁG { is assumed to vary linearly with denaturant concentration and the Gibbs-Helmholtz equation is used to model stability changes with temperature. Approximately 60% of the surface area buried upon folding is buried in the transition state as evidenced by changes in the heat capacity and m value between the unfolded state and the transition state. The equilibrium thermodynamic parameters, ÁC p , m and ÁG , all agree with the values calculated from the kinetic experiments, providing additional evidence that folding is two-state. The folding rates at 0 M guanidine hydrochloride show a non-Arrhenius temperature dependence typical of globular proteins. When the folding rates are examined along constant ÁG /T contours they display an Arrhenius temperature dependence with a slope of À8600 K. This indicates that for this system, the non-Arrhenius temperature dependence of folding can be accounted for by the anomalous temperature dependence of the interactions which stabilize proteins.

Biochemistry, 1989

N M R spectroscopy has been used to investigate the structure of a partially folded state of a pr... more N M R spectroscopy has been used to investigate the structure of a partially folded state of a protein, the molten globule or A-state of a-lactalbumin. The 'H N M R spectrum of this species differs substantially from those of both the native and fully unfolded states, reflecting the intermediate level of order. The resolution in the spectrum is limited by the widespread overlap and substantial line widths of many of the resonances. Methods have therefore been developed that exploit the well-resolved spectrum of the native protein to probe indirectly the A-state. A number of resonances of the A-state have been found to be substantially shifted from their positions in the spectrum of the unfolded state and have been identified through magnetization transfer with the native state, under conditions where the two states are interconverting. The most strongly perturbed residues in the A-state were found to be among those that form a hydrophobic core to the native structure. A number of amides were found to be highly protected from solvent exchange in the A-state. These have been identified through pH-jump experiments, which label them in the spectrum of the native protein. They were found to occur mainly in segments that are helical in the native structure. These results enable a model of the A-state to be proposed in which significant conformational freedom exists but where specific elements of native-like structure are preserved. x e properties of partially organized states of proteins, in which only a subset of the native folding interactions may be present, are of fundamental importance in relation to our understanding of the nature of protein folding. The cooperativity of folding is such that these species often exist only transiently in the course of folding (Tanford, 1968; Kim & Baldwin, 1982), so that their detailed characterization may be feasible only if some form of trapping is possible (Creighton, 1978; Ghelis, 1980; Kim, 1986; Roder, 1988). Recently, however, it has been reported that several proteins can exist, under certain conditions, in partially structured states which +This work was supported by the U.K. Science and Engineering Research Council. J.B. acknowledges receipt of a University of California President's Fellowship and a Fulford Junior Research Fellowship, Somerville College, Oxford. P.A.E. acknowledges an SERC Postdoctoral Fellowship and C.H. a Dee Graduate Scholarship, St. Hugh's College. C.M.D. is a member of the Oxford Centre for Molecular Sciences.

Biochemistry, 1994

The refolding kinetics of hen lysozyme have been studied using a range of fluorescent probes. The... more The refolding kinetics of hen lysozyme have been studied using a range of fluorescent probes. These experiments have provided new insight into the nature of intermediates detected in our recent hydrogenexchange labeling studies [Radford, s. E., et al. (1992) Nature 358,302-3071, which were performed under the same conditions. Protection from exchange results primarily from the development of stabilizing sidechain interactions, and the fluorescence studies reported here have provided a new perspective on this aspect of the refolding process. The intrinsic fluorescence of the six tryptophan residues and its susceptibility to quenching by iodide have been used to monitor the development of hydrophobic structure, and these studies have been complemented by experiments involving binding to a fluorescent hydrophobic dye 1-anilinenaphthalenesulfonic acid (ANS). Formation of fixed tertiary interactions of aromatic residues has been monitored by near-UV circular dichroism, while development of a competent active site has been probed by binding to a competitive inhibitor bearing a fluorescent label, 4-methylumbelliferyl-N,N'-diacetyl-P-We acknowledge support by the Royal Society and by the SERC and MRC through the Oxford Centre for Molecular Sciences and the Cambridge Centre for Molecular Recognition. S.E.R. is a Royal Society 1983 University Research Fellow. C.M.D. is an International Research Scholar of the Howard Hughes Medical Institute.

Folding and Design, 1996

Background: Hydrogen exchange labelling has been a key method in characterizing the structure of ... more Background: Hydrogen exchange labelling has been a key method in characterizing the structure of transient folding intermediates. In studies of several proteins, however, there has been clear spectroscopic evidence for partial folding of some kind at very early times, before any protection from exchange was measurable. These results, presumably a consequence of limited stability of specific backbone interactions, have made it difficult to assess the extent of native-like folding in the very early intermediates. We have used a variant of the labelling method to investigate marginally stable structures formed within the first few milliseconds of refolding of two such proteins, hen lysozyme and ubiquitin. Results: In lysozyme, population of a subset of native-like secondary structures on this timescale is revealed, thus reconciling the exchange behaviour with circular dichroism measurements and confirming the significance of the rapidly formed embryonic structure as a foundation for the subsequent folding pathway. In the case of ubiquitin, by contrast, no significantly protective structure was detectable, suggesting that here secondary structural elements can be populated only marginally ahead of the major cooperative folding event; this was also supported by stopped-flow circular dichroism measurements. Conclusions: The hydrogen exchange approach can be extended to probe the formation of native-like structure formed in very early folding intermediates, even when the stability of specific interactions is marginal. In the case of lysozyme, this has provided a new window on an early stage of organization of the ␣-helical domain.

Folding and Design, 1996

Kinetic studies of folding sometimes reveal very rapid spectroscopic changes that may indicate th... more Kinetic studies of folding sometimes reveal very rapid spectroscopic changes that may indicate the population of intermediates, but it is difficult to elucidate in detail the nature of the interactions involved. In this review, we focus on one important aspect of this problem: how to probe the nature and extent of clustering of hydrophobic sidechains. As the information obtainable from different experimental approaches is outlined, it becomes clear that a combination of methods is likely to be necessary to build up a reasonable picture of early folding events.

Trends in Biochemical Sciences, 1994

Hen lysozyme is one of the best characterized and most studied of all proteins. Recently, we have... more Hen lysozyme is one of the best characterized and most studied of all proteins. Recently, we have used a range of different methods to examine the events involved in the in vitro folding pathway of this protein. In this review we show that, by combining complementary techniques, it has been possible to piece together a detailed model for the folding of this enzyme. Important questions prompted by this work are highlighted and we then propose some ideas consistent with our data, as well as those of others, which we believe begin to provide insight into one of the most intriguing of structural problems in biology--how proteins can achieve their complex native forms from disordered denatured states.

Trends in Biochemical Sciences, 1993

Proteins: Structure, Function, and Bioinformatics, 1992

The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme... more The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30°C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 105–107 times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35°C, by lowering the stability of the native state through selective cleavage of the Cys‐6–Cys‐127 disulfide crosslink (CM6−127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation...

Nature, 1992

Analysis of the folding of hen lysozyme shows that the protein does not become organized in a sin... more Analysis of the folding of hen lysozyme shows that the protein does not become organized in a single cooperative event but that different parts of the structure become stabilized with very different kinetics. In particular, in most molecules the alpha-helical domain folds faster than the beta-sheet domain. Furthermore, different populations of molecules fold by kinetically distinct pathways. Thus, folding is not a simple sequential assembly process but involves parallel alternative pathways, some of which may involve substantial reorganization steps.

Journal of Molecular Biology, 1998

The folding and unfolding kinetics of the N-terminal domain of the ribosomal protein L9 have been... more The folding and unfolding kinetics of the N-terminal domain of the ribosomal protein L9 have been measured at temperatures between 7 and 85 C and between 0 and 6 M guanidine deuterium chloride. Stopped-ow¯uorescence was used to measure rates below 55 C and NMR lineshape analysis was used above 55 C. The amplitudes and rate pro®les of the stopped-¯ow¯uorescence experiments are consistent with a two-state folding mechanism, and plots of ln(k) versus guanidine deuterium chloride concentration show the classic v-shape indicative of two-state folding. There is no roll over in the plots when the experiments are repeated in the presence of 400 mM sodium sulfate. Temperature and denaturant effects were ®t simultaneously to the simple model k D exp(ÀÁG { /RT) where ÁG { represents the change in apparent free energy between the transition state and the folded or unfolded state and D represents the maximum possible folding speed. ÁG { is assumed to vary linearly with denaturant concentration and the Gibbs-Helmholtz equation is used to model stability changes with temperature. Approximately 60% of the surface area buried upon folding is buried in the transition state as evidenced by changes in the heat capacity and m value between the unfolded state and the transition state. The equilibrium thermodynamic parameters, ÁC p , m and ÁG , all agree with the values calculated from the kinetic experiments, providing additional evidence that folding is two-state. The folding rates at 0 M guanidine hydrochloride show a non-Arrhenius temperature dependence typical of globular proteins. When the folding rates are examined along constant ÁG /T contours they display an Arrhenius temperature dependence with a slope of À8600 K. This indicates that for this system, the non-Arrhenius temperature dependence of folding can be accounted for by the anomalous temperature dependence of the interactions which stabilize proteins.

Biochemistry, 1989

N M R spectroscopy has been used to investigate the structure of a partially folded state of a pr... more N M R spectroscopy has been used to investigate the structure of a partially folded state of a protein, the molten globule or A-state of a-lactalbumin. The 'H N M R spectrum of this species differs substantially from those of both the native and fully unfolded states, reflecting the intermediate level of order. The resolution in the spectrum is limited by the widespread overlap and substantial line widths of many of the resonances. Methods have therefore been developed that exploit the well-resolved spectrum of the native protein to probe indirectly the A-state. A number of resonances of the A-state have been found to be substantially shifted from their positions in the spectrum of the unfolded state and have been identified through magnetization transfer with the native state, under conditions where the two states are interconverting. The most strongly perturbed residues in the A-state were found to be among those that form a hydrophobic core to the native structure. A number of amides were found to be highly protected from solvent exchange in the A-state. These have been identified through pH-jump experiments, which label them in the spectrum of the native protein. They were found to occur mainly in segments that are helical in the native structure. These results enable a model of the A-state to be proposed in which significant conformational freedom exists but where specific elements of native-like structure are preserved. x e properties of partially organized states of proteins, in which only a subset of the native folding interactions may be present, are of fundamental importance in relation to our understanding of the nature of protein folding. The cooperativity of folding is such that these species often exist only transiently in the course of folding (Tanford, 1968; Kim & Baldwin, 1982), so that their detailed characterization may be feasible only if some form of trapping is possible (Creighton, 1978; Ghelis, 1980; Kim, 1986; Roder, 1988). Recently, however, it has been reported that several proteins can exist, under certain conditions, in partially structured states which +This work was supported by the U.K. Science and Engineering Research Council. J.B. acknowledges receipt of a University of California President's Fellowship and a Fulford Junior Research Fellowship, Somerville College, Oxford. P.A.E. acknowledges an SERC Postdoctoral Fellowship and C.H. a Dee Graduate Scholarship, St. Hugh's College. C.M.D. is a member of the Oxford Centre for Molecular Sciences.

Biochemistry, 1994

The refolding kinetics of hen lysozyme have been studied using a range of fluorescent probes. The... more The refolding kinetics of hen lysozyme have been studied using a range of fluorescent probes. These experiments have provided new insight into the nature of intermediates detected in our recent hydrogenexchange labeling studies [Radford, s. E., et al. (1992) Nature 358,302-3071, which were performed under the same conditions. Protection from exchange results primarily from the development of stabilizing sidechain interactions, and the fluorescence studies reported here have provided a new perspective on this aspect of the refolding process. The intrinsic fluorescence of the six tryptophan residues and its susceptibility to quenching by iodide have been used to monitor the development of hydrophobic structure, and these studies have been complemented by experiments involving binding to a fluorescent hydrophobic dye 1-anilinenaphthalenesulfonic acid (ANS). Formation of fixed tertiary interactions of aromatic residues has been monitored by near-UV circular dichroism, while development of a competent active site has been probed by binding to a competitive inhibitor bearing a fluorescent label, 4-methylumbelliferyl-N,N'-diacetyl-P-We acknowledge support by the Royal Society and by the SERC and MRC through the Oxford Centre for Molecular Sciences and the Cambridge Centre for Molecular Recognition. S.E.R. is a Royal Society 1983 University Research Fellow. C.M.D. is an International Research Scholar of the Howard Hughes Medical Institute.

Folding and Design, 1996

Background: Hydrogen exchange labelling has been a key method in characterizing the structure of ... more Background: Hydrogen exchange labelling has been a key method in characterizing the structure of transient folding intermediates. In studies of several proteins, however, there has been clear spectroscopic evidence for partial folding of some kind at very early times, before any protection from exchange was measurable. These results, presumably a consequence of limited stability of specific backbone interactions, have made it difficult to assess the extent of native-like folding in the very early intermediates. We have used a variant of the labelling method to investigate marginally stable structures formed within the first few milliseconds of refolding of two such proteins, hen lysozyme and ubiquitin. Results: In lysozyme, population of a subset of native-like secondary structures on this timescale is revealed, thus reconciling the exchange behaviour with circular dichroism measurements and confirming the significance of the rapidly formed embryonic structure as a foundation for the subsequent folding pathway. In the case of ubiquitin, by contrast, no significantly protective structure was detectable, suggesting that here secondary structural elements can be populated only marginally ahead of the major cooperative folding event; this was also supported by stopped-flow circular dichroism measurements. Conclusions: The hydrogen exchange approach can be extended to probe the formation of native-like structure formed in very early folding intermediates, even when the stability of specific interactions is marginal. In the case of lysozyme, this has provided a new window on an early stage of organization of the ␣-helical domain.