Intrinsically disordered proteins (IDPs) do not form stable tertiary structures like their ordered partners. They exist as heterogeneous ensembles that fluctuate over a time scale. Intrinsically disordered regions and proteins are found across different phyla and exert crucial biological functions. They exhibit transient secondary structures in their free state and become folded upon binding to their protein partners via a mechanism called coupled folding and binding. Some IDPs form alpha helices when bound to their protein partners. We observed a set of cancer associated IDPs where the helical binding segments of IDPs are flanked by prolines on both the sides. Helix-breaking prolines are frequently found in IDPs flanking the binding segment and are evolutionarily conserved across phyla. Two studies have shown that helix flanking prolines modulate the function of IDPs by regulating the levels of disorder in their free state and in turn regulating the binding affinities to their partners. We aimed to study if this is a common phenomenon in IDPs that exhibit similar pattern in the conservation of helix flanking prolines. We chose to test the hypothesis in c-Myb-KIX : IDP-target system in which the disordered protein exhibits high residual helicity levels in its free state. c-Myb is a hematopoietic regulator that plays a crucial role in cancer by binding to the KIX domain of CBP. Studying the functional regulation of c-Myb by modulating the disorder levels in c-Myb and in IDPs in general provides a better understanding of the way IDPs function and can be used in therapeutic strategies as IDPs are known to be involved in regulating various cellular processes and diseases. To study the effect of conserved helix flanking prolines on the residual helicity levels of c-Myb and its binding affinity to the KIX domain of CBP, we mutated the prolines to alanines. Mutating prolines to alanines increased the helicity levels of c-Myb in its free state. This small increase in the helicity levels of a highly helical c-Myb showed almost no effect on the binding affinity between cMyb and KIX. We hypothesized that there is a helical threshold for coupled folding and binding beyond which helicity levels of the free state IDP have no effect on its binding to their ordered protein partner. To test this hypothesis, we mutated solvent exposed amino acid residues in c-Myb that reduce its overall helicity and studied its effect on the binding affinity between c-Myb and KIX. Over a broad range of reduction in helicity levels of the free state did not show an effect on the binding affinity but beyond a certain level, decrease in helicity levels showed pronounced effects on the binding affinity between c-Myb and KIX.

Decades have passed since the realisation that a protein’s amino acid sequence can contain all the information required to form a complex three-dimensional fold. Until recently, these encoded structures were thought to be crucial determinants of protein function. Much effort was directed to fully understand the mechanisms behind how and why proteins fold, with natively unfolded proteins thought to be experimental artefacts. Today, the field of natively unfolded – or so-called intrinsically disordered – proteins, is rapidly developing. Protein disorder content has been positively correlated with organismal complexity, with over thirty percent of eukaryotic proteins predicted to contain disordered regions. However, the biophysical consequences of disorder are yet to be fully determined. With the aim of addressing some of the outstanding questions, the work described in this thesis focuses on the relevance of structure within disordered proteins. Whilst populating a variety of conformations in isolation, a subset of disordered proteins can fold upon binding to a partner macromolecule. This folded state may be present within the ensemble of conformations sampled by the unbound protein, opening the question of what comes first: folding or binding? Protein engineering techniques were employed to alter the level of residual ‘bound-like’ structure within the free conformational ensemble, and the consequences on coupled folding and binding reactions were investigated. Resultant changes in the rate of association are easily imaginable; yet, this work demonstrates that the majority of the observed changes in binding affinity were due to alterations in the rate of dissociation, thus altering the lifetime of the bound complex. Promiscuous binding is a touted advantage of being disordered. If many disordered proteins, each with their own conformational ensemble, can bind and fold to the same partner, then where is the folding component encoded? Does the partner protein template the folding reaction? Or, is the folding information contained within the disordered protein sequence? Utilising phi-value analysis on the BCL-2 family of proteins, residues in the disordered sequence were probed to ascertain which form contacts at the transition state of the reaction. Comparison with phi-value analyses of alternative pairs – sharing either the ordered or disordered protein – provides insight into the encoding of these interactions. In the context of a bimolecular reaction, the amino acid sequence of the disordered protein was shown to determine the interactions within the transition state. Thus, analogous to the discovery from decades’ past, it is the sequence of the protein that folds which encodes its pathway, even when binding is a prerequisite.

Proteins are synthesised as linear polymeric chains. The subtle energetic interplay of interatomic interactions results in chain folding, through which proteins may acquire defined structures. This spatial organisation is encoded by the protein sequence itself; the so-called thermodynamic hypothesis formulated by Anfinsen in 1961. A defined structure is often considered a pre-requisite to protein function, but widespread existence of intrinsically disordered proteins (IDPs) has prompted a re- evaluation of the ways biological function may be encoded into polypeptide chains. Furthermore, proteins often exist as part of multi-component entities, where regulation of assembly is integral to their properties. The interplay between disorder, oligomerisation and function is the focus of this thesis. Some IDPs fold conditionally upon interacting with a partner protein; a process known as coupled folding and binding. What are the biophysical advantages and consequences of disorder in the context of these interactions? A common feature of IDPs is their sequence composition bias, with charged residues being often over-represented. It is therefore tempting to speculate that electrostatic interactions may play a major role in coupled folding and binding reactions. Surprisingly, the opposite was found to be true. Charge-charge interactions only contributed about an order of magnitude to the association rate constants of two contrasting model systems. The lack of pre-formed binding interfaces-a consequence of disorder-might preclude electrostatic acceleration from complementary patches. By looking at the role of the sequence, many studies have taken a protein-centric approach to understanding disorder. Yet there is paucity of data about the effect of extrinsic factors on interactions involving disordered partners. Investigating the role of co-solutes, it was discovered that the kinetic and thermodynamic profiles of coupled folding and binding reactions were sensitive to ion-types. This effect followed the Hofmeister series, and occurred at physiological concentrations of salt. The sensitivity of coupled folding and binding reactions-a consequence of the lack of stability of IDPs-might be advantageous. Given the role of ions in biology, this 'biophysical sensing' could be a mechanism of physiological relevance, allowing modulation of protein-protein interactions involving disordered partners in response to changes in their environments. In cells, signalling networks are often multi-layered, and involve competing protein-protein interactions. The interplay between the biophysical characteristics of the components, and the behaviour of the network were investigated in a model tripartite system composed of folded and disordered proteins. The BCL-2 family regulates the intrinsic pathway of apoptosis through control of mitochondrial outer-membrane permeabilisation; a result of BAK and BAX oligomerisation. Through a shared homology motif (termed BH3), the subtle balance of their interactions determines cellular fate at the molecular level. Characterisation of the model under simple biochemical conditions revealed large differences in affinities among binary interactions; the consequence of the lifetime of the complexes, not their speed of association. A membrane-like environment, re-created using detergents, allows the oligomerisation of BAK and BAX in vitro. Furthermore, investigation of the tripartite system under detergent conditions showed that regulation of the network was the result of competing hetero- and homo-oligomerisation events. Relationships to their biophysical properties were gained by probing their energy landscapes using protein folding techniques. The connection between the biophysical properties of the components of the network and their interactions provides a molecular explanation for the regulation of apoptosis. This thesis offers insights into the ways structured assemblies and environmentally responsive disorder elements may encode functions into proteins.

The interaction between two 6 kDa proteins has been investigated. The studied complex of micromolar affinity (Kd) consists of the Z domain derived from staphylococcal protein A and the related protein ZSPA-1, belonging to a group of binding proteins denoted affibody molecules generated via combinatorial engineering of the Z domain. Affibody-target protein complexes are good model systems for structural and thermodynamic studies of protein-protein interactions. With the Z:ZSPA-1 pair as a starting point, we determined the solution structure of the complex and carried out a preliminary characterization of ZSPA-1. We found that the complex contains a rather large (ca. 1600 Å2) interaction interface with tight steric and polar/nonpolar complementarity. The structure of ZSPA-1 in the complex is well-ordered in a conformation that is very similar to that of the Z domain. However, the conformation of the free ZSPA-1 is best characterized by comparisons with protein molten globules. It shows a reduced secondary structure content, aggregation propensity, poor thermal stability, and binds the hydrophobic dye ANS. This molten globule state of ZSPA-1 is the native state in the absence of the Z domain, and the ordered state is only adopted following a stabilization that occurs upon binding. A more extensive characterization of ZSPA-1 suggested that the average topology of the Z domain is retained in the molten globule state but that it is represented by a multitude of conformations. Furthermore, the molten globule state is only marginally stable, and a significant fraction of ZSPA-1 exists in a completely unfolded state at room temperature. A complete thermodynamic characterization of the Z:ZSPA-1 pair suggests that the stabilization of the molten globule state to an ordered three helix structure in the complex is associated with a significant conformational entropy penalty that might influence the binding affinity negatively and result in an intermediate-affinity (µM) binding protein. This can be compared to a dissociation constant of 20-70 nM for the complex Z:Fc of IgG where Z uses the same binding surface as in Z:ZSPA-1. Structure analyses of Z in the free and bound state reveal an induced fit response upon complex formation with ZSPA-1 where a conformational change of several side chains in the binding surface increases the accessible surface area with almost 400 Å2 i.e. almost half of the total interaction surface in the complex. Two cysteine residues were introduced at specific positions in ZSPA-1 for five mutants in order to stabilize the conformation of ZSPA-1 by disulfide bridge formation. The mutants were thermodynamically characterized and the binding affinity of one mutant showed an improvement by more than a factor of ten. The improvement of the introduced cysteine bridge correlates with an increase in binding enthalpy rather than with entropy. Further analysis of the binding entropy suggests that the conformational entropy change in fact is reduced but its favorable contribution is opposed by a less favorable desolvation enthalpy change. These studies illustrate the structural and thermodynamic complexity of protein-protein interactions, but also that this complexity can be dissected and understood. In this study, a comprehensive characterization of the ZSPA-1 affibody has gained insight into the intricate mechanisms involved in complex formation. These theories were supported by the design of a ZSPA-1 mutant with improved binding affinity. / QC 20100924