Philip A. Evans

Understanding how proteins fold: the lysozyme story so far.

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.

Structure of very early protein folding intermediates: new insights through a variant of hydrogen exchange labelling

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 alpha-helical domain.

Probing the structure of folding intermediates

Abstract The description of protein folding mechanisms in terms of the structures of well defined, partially folded intermediates is a major objective in structural biology. Despite the fleeting existence of intermediates and the fact that their structures are unlikely to be uniquely defined, significant advances in our understanding of protein folding pathways have been made in the last year based on a variety of physical methods.

Proton NMR studies of denatured lysozyme.

Evidence is presented from 1H NMR studies for non‐random conformational behaviour in denatured lysozyme in aqueous solution. A method is presented which permits the assignment of resonances in the 1H NMR spectrum of the denatured protein by observing magnetisation transfer from resonances of the native state. The use of these experiments in characterising the denatured state and the significance of these studies for the investigation of protein folding are discussed.

A peptide model for proline isomerism in the unfolded state of staphylococcal nuclease.

Nuclear magnetic resonance spectroscopy has been used to investigate a synthetic peptide (YVYKPNNTHE) corresponding to residues 113 to 122 of staphylococcal nuclease. In the major folded state of the protein this region forms a type VIa beta-turn containing a cis Lys116-Pro117 peptide bond. There is, however, no evidence for any significant population of such a turn in the peptide in aqueous solution and the X-Pro bond is predominantly in the trans configuration. The peptide exhibits several well-resolved minor resonances due to the presence of a small fraction (4 +/- 2%) of the cis-proline isomer. The ratio of cis to trans isomer populations was found to be independent of temperature between 5 degrees C and 70 degrees C, indicating that delta H for the isomerism is close to zero. Using magnetization transfer techniques the rate of trans to cis interconversion was found to be 0.025(+/- 0.013) s-1 at 50 degrees C. The thermodynamics and kinetics of isomerism in the peptide are very similar to those estimated for the Lys116-Pro117 peptide bond in unfolded nuclease, suggesting that the cis-trans equilibrium in the unfolded protein is largely determined by the residues adjacent to Pro117 in the sequence. These results are consistent with previous suggestions that the cis-proline bond is stabilized late in the folding process and that the predominance of the cis form in folded nuclease is due to stabilizing interactions within the protein that give rise to a favorable enthalpy term.

The role of a β-bulge in the folding of the β-hairpin structure in ubiquitin

It is known that the peptide corresponding to the N‐terminal β‐hairpin of ubiquitin, U(1–17), can populate the monomeric β‐hairpin conformation in aqueous solution. In this study, we show that the Gly‐10 that forms the bulge of the β‐turn in this hairpin is very important to the stability of the hairpin. The deletion of this residue to desG10(1–16) unfolds the structure of the peptide in water. Even under denaturing conditions, this bulge appears to be important in maintaining the residual structure of ubiquitin, which involves tertiary interactions within the sequence 1 to 34 in the denatured state. We surmise that this residual structure functions as one of the nucleation centers in the folding process and is important in stabilizing the transition state. In accordance with this idea, deleting Gly‐10 slows down the refolding and unfolding rate by about one half.

Experimental investigation of sidechain interactions in early folding intermediates

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.

Proton NMR Studies of Protein Dynamics and Folding: Applications of Magnetization Transfer NMR

When a protein exists at equilibrium in more than one conformational state it may be possible to observe separately in the NMR spectrum resonances corresponding to the different states. Provided that interconversion between these states occurs at suitable rates, magnetization transfer techniques may be used to detect it, and in favorable cases to obtain rate constants for specific conformational transitions. Results of one- and two-dimensional 1H NMR experiments with lysozyme and staphylococcal nuclease are used to illustrate the potentials of such an approach to studying the dynamics and folding of proteins.

TWO-DIMENSIONAL EXCHANGE EXPERIMENTS IN NMR STUDIES OF PROTEIN DYNAMICS AND FOLDING

ABSTRACT The application of two-dimensional nmr spectroscopy to the study of the kinetics of molecular conformational transitions is discussed. Results of experiments with lysozyme and staphylococcal nuclease are used to illustrate the potential of such an approach to studying the dynamics and folding of proteins.