Allostery is the process by which biological macromolecules (mostly proteins) transmit the effect of binding at one site to another, often distal, functional site, allowing for regulation of activity. Recent experimental observations demonstrating that allostery can be facilitated by dynamic and intrinsically disordered proteins have resulted in a new paradigm for understanding allosteric mechanisms, which focuses on the conformational ensemble and the statistical nature of the interactions responsible for the transmission of information. Analysis of allosteric ensembles reveals a rich spectrum of regulatory strategies, as well as a framework to unify the description of allosteric mechanisms from different systems.

The heat capacity plays a major role in the determination of the energetics of protein folding and molecular recognition. As such, a better understanding of this thermodynamic parameter and its structural origin will provide new insights for the development of better molecular design strategies. In this paper we have analyzed the absolute heat capacity of proteins in different conformations. The results of these studies indicate that three major terms account for the absolute heat capacity of a protein: (1) one term that depends only on the primary or covalent structure of a protein and contains contributions from vibrational frequencies arising from the stretching and bending modes of each valence bond and internal rotations; (2) a term that contains the contributions of noncovalent interactions arising from secondary and tertiary structure; and (3) a term that contains the contributions of hydration. For a typical globular protein in solution the bulk of the heat capacity at 25 degrees C is given by the covalent structure term (close to 85% of the total). The hydration term contributes about 15 and 40% to the total heat capacity of the native and unfolded states, respectively. The contribution of non-covalent structure to the total heat capacity of the native state is positive but very small and does not amount to more than 3% at 25 degrees C. The change in heat capacity upon unfolding is primarily given by the increase in the hydration term (about 95%) and to a much lesser extent by the loss of noncovalent interactions (up to approximately 5%).(ABSTRACT TRUNCATED AT 250 WORDS)

Transcription factors and other allosteric cell signaling proteins contain a disproportionate number of domains or segments that are intrinsically disordered (ID) under native conditions. In many cases folding of these segments is coupled to binding with one or more of their interaction partners, suggesting that intrinsic disorder plays an important functional role. Despite numerous hypotheses for the role of ID domains in regulation, a mechanistic model has yet to be established that can quantitatively assess the importance of intrinsic disorder for intramolecular site-to-site communication, the hallmark property of allosteric proteins. Here, we present such a model and show that site-to-site allosteric coupling is maximized when intrinsic disorder is present in the domains or segments containing one or both of the coupled binding sites. This result not only explains the prevalence of ID domains in regulatory proteins, it also calls into question the classical mechanical view of energy propagation in proteins, which predicts that site-to-site coupling would be maximized when a well defined pathway of folded structure connects the two sites. Furthermore, in showing that the coupling mechanism conferred by intrinsic disorder is robust and independent of the network of interactions that physically link the coupled sites, unique insights are gained into the energetic ground rules that govern site-to-site communication in all proteins.allostery ͉ ensemble ͉ regulation ͉ site-to-site communication O ver the past decade, the paradigm that proteins function by adopting highly ordered structures has been challenged by the observation that thousands of different proteins are likely to be intrinsically disordered (i.e., sample multiple conformations) or have intrinsically disordered (ID) domains under native conditions (1-8). Of particular significance is the growing body of evidence that intrinsic disorder is found in disproportionately higher amounts in cell signaling proteins and transcription factors, suggesting an important role in their regulatory capacity (5). Indeed for cases where detailed study has been performed, structure formation in these proteins (or domains) is linked to ligand binding in other parts of the molecule, indicating that the order/disorder transition is coupled to long-range allosteric communication within the molecule and is therefore important to its functional role (5). Interestingly, within specific classes of regulatory proteins [such as the steroid hormone receptors (9)], functionally analogous domains relevant to transcription regulation appear to be ID in each member, even though there is little sequence conservation in these regions. This finding suggests that the underlying regulatory mechanism is both effective and robustly encoded in nature.Hypotheses for the role of intrinsic disorder include: highspecificity/low-affinity binding (3, 5), rapid protein turnover (1), and high specificity for multiple targets (1-5). However, despite the clear experimental evidence for the existence of...

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Allostery is a biological phenomenon of fundamental importance in regulation and signaling, and efforts to understand this process have led to the development of numerous models. In spite of individual successes in understanding the structural determinants of allostery in well-documented systems, much less success has been achieved in identifying a set of quantitative and transferable ground rules that provide an understanding of how allostery works. Are there organizing principles that allow us to relate structurally different proteins, or are the determinants of allostery unique to each system? Using an ensemble-based model, we show that allosteric phenomena can be formulated in terms of conformational free energies of the cooperative elements in a protein and the coupling interactions between them. Interestingly, the resulting allosteric ground rules provide a framework to reconcile observations that challenge purely structural models of site-to-site coupling, including (a) allostery in the absence of pathways of structural distortions, (b) allostery in the absence of any structural change, and (c) the ability of allosteric ligands to act as agonists under some circumstances and antagonists under others. The ensemble view of allostery that emerges provides insights into the energetic prerequisites of site-to-site coupling and thus into how allostery works.

Allosteric regulation plays an important role in many biological processes, such as signal transduction, transcriptional regulation, and metabolism. Allostery is rooted in the fundamental physical properties of macromolecular systems, but its underlying mechanisms are still poorly understood. A collection of contributions to a recent interdisciplinary CECAM (Center Européen de Calcul Atomique et Moléculaire) workshop is used hereto provide an overview of the progress and remaining limitations in the understanding of the mechanistic foundations of allostery gained from computational and experimental analyses of real protein systems and model systems. The main conceptual frameworks instrumental in driving the field are discussed. We illustrate the role of these frameworks in illuminating molecular mechanisms and explaining cellular processes, and describe some of their promising practical applications in engineering molecular sensors and informing drug design efforts.

To explore how distal mutations affect binding sites and how binding sites in proteins communicate, an ensemble-based model of the native state was used to define the energetic connectivities between the different structural elements of Escherichia coli dihydrofolate reductase. Analysis of this model protein has allowed us to identify two important aspects of intramolecular communication. First, within a protein, pair-wise couplings exist that define the magnitude and extent to which mutational effects propagate from the point of origin. These pair-wise couplings can be identified from a quantity we define as the residue-specific connectivity. Second, in addition to the pair-wise energetic coupling between residues, there exists functional connectivity, which identifies energetic coupling between entire functional elements (i.e., binding sites) and the rest of the protein. Analysis of the energetic couplings provides access to the thermodynamic domain structure in dihydrofolate reductase as well as the susceptibility of the different regions of the protein to both small-scale (e.g., point mutations) and large-scale perturbations (e.g., binding ligand). The results point toward a view of allosterism and signal transduction wherein perturbations do not necessarily propagate through structure via a series of conformational distortions that extend from one active site to another. Instead, the observed behavior is a manifestation of the distribution of states in the ensemble and how the distribution is affected by the perturbation. M ost cellular processes, which are facilitated by proteins, are modulated by effectors. The basic features of such a mechanism of regulation are the presence of multiple binding sites for various ligands and communication between these binding sites, which often are situated many angstroms apart. An understanding of the ground rules of this regulatory mechanism requires a quantitative definition of the functional linkages between these binding sites. In 1964, Wyman introduced the thermodynamic concept of linked functions to establish a quantitative formulation for describing the mutual influence of binding sites on each other (1). Linkage theory is based on thermodynamic principles, is applicable to all biological systems, and exhibits quantitative predictive power. Although Wyman's theory provides the mathematical relationships, it does not address the mechanism through which different binding sites communicate. Thus, besides functional linkage, there are underlying structural-thermodynamic linkages that define the mechanism of site-site communication.Despite a significant body of literature showing that information is transmitted through biological systems via a series of interand intramolecular communication events (refs. 2-5 and references therein), a quantitative predictive theory of structural linkage analogous to the Wyman Linkage Theory is not available. Recently, however, a theoretical approach was established to treat the native state of a protein as an ensemble of conformationa...

Cooperative interactions link the behavior of different amino acid residues within a protein molecule. As a result, the effects of chemical or physical perturbations to any given residue are propagated to other residues by an intricate network of interactions. Very often, amino acids ''sense'' the effects of perturbations occurring at very distant locations in the protein molecule. In these studies, we have investigated by computer simulation the structural distribution of those interactions. We show here that cooperative interactions are not intrinsically bi-directional and that different residues play different roles within the intricate network of interactions existing in a protein. The effect of a perturbation to residue j on residue k is not necessarily equal to the effect of the same perturbation to residue k on residue j. In this paper, we introduce a computer algorithm aimed at mapping the network of cooperative interactions within a protein. This algorithm exhaustively performs single site thermodynamic mutations to each residue in the protein and examines the effects of those mutations on the distribution of conformational states. The algorithm has been applied to three different proteins ( repressor fragment 6-85, chymotrypsin inhibitor 2, and barnase). This algorithm accounts well for the observed behavior of these proteins.Protein folding is a highly cooperative process. One of the most notable manifestations of cooperativity is that the vast majority of conformational states that are accessible to a protein have a negligible probability and never become populated to a significant extent. In most situations, the folding͞unfolding equilibrium is well accounted for by a two-state process in which the population of intermediates is assumed to be zero (1). Despite this fact, there is ample evidence, particularly from hydrogen exchange data obtained under native conditions, that some partially folded conformations are always present. The observed heterogeneity in the magnitude of the hydrogen exchange protection factors measured under native conditions indicates that certain residues become exposed to the solvent as a result of local rather than global unfolding (2-13). If this is the case, cooperative interactions do not involve the entire protein molecule, and the conformational equilibrium cannot be considered as an all-or-none process in which the entire protein is either folded or unfolded. If cooperative interactions do not extend uniformly throughout the entire protein molecule, then some residues will have a more important role than others in defining cooperativity. The purpose of this paper is to identify those residues and investigate the structural distribution of cooperative interactions in proteins.From a rigorous point of view, cooperativity originates when the partition function of a system cannot be written as the product of the individual partition functions of the constituent subsystems. This situation occurs when the interaction energy among different subsystems is not zero. In...