Alan R. Fersht

The folding of an enzyme: I. Theory of protein engineering analysis of stability and pathway of protein folding

The theory, assumptions and limitations are outlined for a simple protein engineering approach to the problem of the stability and pathway of protein folding. It is a general procedure for analysing structure-activity relationships in non-covalent bonding, including enzyme catalysis, that relates experimentally accessible data to changes in non-covalent bonding. Kinetic and equilibrium measurements on the unfolding and refolding of mutant proteins can be used to map the formation of structure in transition states and folding intermediates. For example, the ratio of the changes in the activation energy of unfolding and the free energy of unfolding on mutation is measured to give a parameter phi. There are two extreme values of phi that are often found in practice and may be interpreted in a simple manner. A value of phi = 0 implies that the structure at the site of mutation is as folded in the transition state as it is in the folded state. Conversely, phi = 1 shows that the structure at the site of mutation is as unfolded in the transition state as it is in the unfolded structure. Fractional values of phi are more difficult to interpret and require a more sophisticated approach. The most suitable mutations involve truncation of side-chains to remove moieties that preferably make few interactions with the rest of the protein and do not pair with buried charges. Fractional values of phi found for this type of mutation may imply that there is partial non-covalent bond formation or a mixture of states. The major assumptions of the method are: (1) mutation does not alter the pathway of folding; (2) mutation does not significantly change the structure of the folded state; (3) mutation does not perturb the structure of the unfolded state; and (4) the target groups do not make new interactions with new partners during the course of reaction energy. Assumptions (2) and (3) are not necessarily essential for the simple cases of phi = 0 or 1, the most common values, since effects of disruption of structure can cancel out. Assumption (4) may be checked by the double-mutant cycle procedure, which may be analysed to isolate the effects of just a pair of interactions against a complicated background. This analysis provides the formal basis of the accompanying studies on the stability and pathway of folding of barnase, where it is seen that the theory holds very well in practice.

Awakening guardian angels: drugging the p53 pathway

Currently, around 11 million people are living with a tumour that contains an inactivating mutation of TP53 (the human gene that encodes p53) and another 11 million have tumours in which the p53 pathway is partially abrogated through the inactivation of other signalling or effector components. The p53 pathway is therefore a prime target for new cancer drug development, and several original approaches to drug discovery that could have wide applications to drug development are being used. In one approach, molecules that activate p53 by blocking protein–protein interactions with MDM2 are in early clinical development. Remarkable progress has also been made in the development of p53-binding molecules that can rescue the function of certain p53 mutants. Finally, cell-based assays are being used to discover compounds that exploit the p53 pathway by either seeking targets and compounds that show synthetic lethality with TP53 mutations or by looking for non-genotoxic activators of the p53 response.

The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus)

In a previous study, a mutant of tyrosyl-tRNA synthetase in which a threonine residue (Thr51) was converted to proline dramatically improved the affinity of the enzyme for its ATP substrate. How does Pro51 improve the enzymes affinity for ATP? A priori, Pro51 might interact directly with the ATP, or it might distort the polypeptide backbone and thereby force new or improved contacts elsewhere from the enzyme to ATP. By making mutants of the Pro51 enzyme at two residues that make hydrogen bonds to the ATP substrate, we show that Pro51 greatly improves the strength of one of these contacts. Thus the propagation of a structural change in an enzyme induced by mutation may be detected by the introduction of further mutations.

Nucleation mechanisms in protein folding

Experiment and theory are converging on the importance of nucleation mechanisms in protein folding. These mechanisms do not use classic nuclei, which are well formed elements of structure present in ground states, but they use diffuse, extended regions, which are observed in transition states.

Energetics of protein-protein interactions: Analysis ofthe Barnase-Barstar interface by single mutations and double mutant cycles

The interaction of barnase, an extracellular RNase of Bacillus amylolique-faciens, with its intracellular inhibitor barstar is a suitable paradigm for protein-protein interactions, since the structures of both the free and the complexed proteins are available at high resolution. The contributions of residues from both proteins to the energetics of kinetics and thermodynamics of binding were measured by double mutant cycle analysis. Such cycles reveal whether the contributions from a pair of residues are additive, or the effects of mutations are coupled. The aim of the study was to determine which of the interactions are co-operative. Double mutant cycles were constructed between a subset of five barnase and seven barstar residues, which were shown by structural and mutagenesis studies to be important in stabilising the complex. The coupling energy between two residues was found to decrease with the distance between them. Generally, residues separated by less than 7 A interact co-operatively. At greater separations, the effects of mutation are additive, and the energetics of the interactions are independent of each other. The highest coupling energies are found between pairs of charged residues (1.6 to 7 kcal mol-1). Three of the six most important interactions detected by double mutant cycle analysis (with coupling energies of more than 3.0 kcal mol-1) had not been noted previously from examination of the crystal structure. The effects of mutation on the kinetics of association are all additive, apart from charged residues located at distances of up to 10 A apart, which are co-operative. This can be explained by the fact that the transition state for association occurs before most interactions are formed.

Rescuing the function of mutant p53

One protein — p53 — plays nemesis to most cancers by condemning damaged cells to death or quarantining them for repair. But the activity of p53 relies on its intact native conformation, which can be lost following mutation of a single nucleotide. With thousands of such mutations identified in patients, how can a future cancer drug buttress this fragile protein structure and restore the cells natural defence?

Protein Folding and Unfolding at Atomic Resolution

Experiment and simulation are now conspiring to give atomic-level descriptions of protein folding relevant to folding, misfolding, trafficking, and degradation in the cell. We are on the threshold of predicting those protein folding events using simulation that has been carefully benchmarked by experiment.

The complete folding pathway of a protein from nanoseconds to microseconds

Combining experimental and simulation data to describe all of the structures and the pathways involved in folding a protein is problematical. Transition states can be mapped experimentally by φ values, but the denatured state is very difficult to analyse under conditions that favour folding. Also computer simulation at atomic resolution is currently limited to about a microsecond or less. Ultrafast-folding proteins fold and unfold on timescales accessible by both approaches, so here we study the folding pathway of the three-helix bundle protein Engrailed homeodomain. Experimentally, the protein collapses in a microsecond to give an intermediate with much native α-helical secondary structure, which is the major component of the denatured state under conditions that favour folding. A mutant protein shows this state to be compact and contain dynamic, native-like helices with unstructured side chains. In the transition state between this and the native state, the structure of the helices is nearly fully formed and their docking is in progress, approximating to a classical diffusion–collision model. Molecular dynamics simulations give rate constants and structural details highly consistent with experiment, thereby completing the description of folding at atomic resolution.

Structural biology of the tumor suppressor p53.

The tumor suppressor protein p53 induces or represses the expression of a variety of target genes involved in cell cycle control, senescence, and apoptosis in response to oncogenic or other cellular stress signals. It exerts its function as guardian of the genome through an intricate interplay of independently folded and intrinsically disordered functional domains. In this review, we provide insights into the structural complexity of p53, the molecular mechanisms of its inactivation in cancer, and therapeutic strategies for the pharmacological rescue of p53 function in tumors. p53 emerges as a paradigm for a more general understanding of the structural organization of modular proteins and the effects of disease-causing mutations.

Is there a unifying mechanism for protein folding

Proteins appear to fold by diverse pathways, but variations of a simple mechanism - nucleation-condensation - describe the overall features of folding of most domains. In general, secondary structure is inherently unstable and its stability is enhanced by tertiary interactions. Consequently, an extensive interplay of secondary and tertiary interactions determines the transition-state for folding, which is structurally similar to the native state, being formed in a general collapse (condensation) around a diffuse nucleus. As the propensity for stable secondary structure increases, folding becomes more hierarchical and eventually follows a framework mechanism where the transition state is assembled from pre-formed secondary structural elements.