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...

The activation domains of many transcription factors appear to exist naturally in an unfolded or only partially folded state. This seems to be the case for AF1/tau1, the major transactivation domain of the human glucocorticoid receptor. We show here that in buffers containing the natural osmolyte trimethylamine N-oxide (TMAO), recombinant AF1 folds into more a compact structure, as evidenced by altered fluorescence emission, circular dichroism spectra, and ultracentrifugal analysis. This conformational transition is cooperative, a characteristic of proteins folding to natural structures. The structure resulting from incubation in TMAO causes the peptide to resist proteolysis by trypsin, chymotrypsin, endoproteinase Arg-C and endoproteinase Gluc-C. Ultracentrifugation studies indicate that AF1/tau1 exists as a monomer in aqueous solution and that the presence of TMAO does not lead to oligomerization or aggregation. It has been suggested that recombinant AF1 binds both the ubiquitous coactivator CBP and the TATA boxbinding protein, TBP. Interactions with both of these are greatly enhanced in the presence of TMAO. Co-immunoadsorption experiments indicate that in TMAO each of these and the coactivator SRC-1 are found complexed with AF1. These data indicate that TMAO induces a conformation in AF1/tau1 that is important for its interaction with certain co-regulatory proteins.

Amino acid substitutions at distant sites in the Escherichia coli cyclic AMP receptor protein (CRP) have been shown to affect both the nature and magnitude of the energetics of cooperativity of cAMP binding, ranging from negative to positive. In addition, the binding to DNA is concomitantly affected. To correlate the effects of amino acid substitutions on the functional energetics and global structural properties in CRP, the partial specific volume (v(o)), the coefficient of adiabatic compressibility (beta(s)(o)), and the rate of amide proton exchange were determined for the wild-type and eight mutant CRPs (K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L) by using sound velocity, density measurements, and hydrogen-deuterium exchange as monitored by Fourier transform infrared spectroscopy at 25 degrees C. These mutations induced large changes in v(o) (0.747-0.756 mL/g) and beta(s)(o) (6.89-9.68 Mbar(-1)) compared to the corresponding values for wild-type CRP (v(o)= 0.750 mL/g and beta(s)(o)= 7.98 Mbar(-1)). These changes in global structural properties correlated with the rate of amide proton exchange. A linear correlation was established between beta(s)(o) and the energetics of cooperativity of binding of cAMP to the high-affinity sites, regardless of the nature of cooperativity, be it negative or positive. This linear correlation indicates that the nature and magnitude of cooperativity are a continuum. A similar linear correlation was established between compressibility and DNA binding affinity. In addition, linear correlations were also found among the dynamics of CRP and functional energetics. Double mutation (K52N/H159L) at positions 52 and 159, whose alpha-carbons are separated by 34.6 A, showed nonadditive effects on v(o) and beta(s)(o). These results demonstrate that a small alteration in the local structure due to amino acid substitution is dramatically magnified in the overall protein dynamics which plays an important role in modulating the allosteric behavior of CRP.

Studies of individual domains or subdomains of the proteins making up the nuclear receptor family have stressed their modular nature. Nevertheless, these receptors function as complete proteins. Studies of specific mutations suggest that in the holoreceptors, intramolecular domain-domain interactions are important for complete function, but there is little knowledge concerning these interactions. The important transcriptional transactivation function in the N-terminal part of the glucocorticoid receptor (GR) appears to have little inherent structure. To study its interactions with the DNA binding domain (DBD) of the GR, we have expressed the complete sequence from the N-terminal through the DBD of the human GR. Circular dichroism analyses of this highly purified, multidomain protein show that it has a considerable helical content. We hypothesized that binding of its DBD to the cognate glucocorticoid response element would confer additional structure upon the N-terminal domain. Circular dichroism and fluorescence emission studies suggest that additional helicity as well as tertiary structure occur in the two-domain protein upon DNA binding. In sum, our data suggest that interdomain interactions consequent to DNA binding imparts structure to the portion of the GR that contains a major transactivation domain.The major identified domains of the nuclear family of receptors are those for ligand binding, DNA binding (DBD), 1 and transactivation, with other functional areas mapped throughout the molecule (1-4). Although the ligand and DNA binding domains have modular structures and, in "domain swapping" experiments, a remarkable ability to carry out their function within the context of other proteins, intramolecular signaling is also important for the proper natural functions of these receptors. Recent experiments studying the effect of mutations on function emphasize the importance of this intramolecular signaling (5, 6). How and when information is exchanged between domains is largely unknown. This is due in part to the fact that no structures are yet available for multidomain proteins from the nuclear receptor family. One intriguing problem is the structural basis for the major transcriptional transactivation function (AF1, tau1) that mutagenesis experiments have defined in the human GR (7). By molecular genetics, AF1 is defined by amino acids 77-262. It appears to function by evoking physical interactions with the basal transcription mechanism, including Ada2 and TATA-binding protein (TBP), possibly through intermediary adapter protein (8, 9). But unlike the ligand binding domain and DBD, AF1 does not appear to function well out of its protein context. Studies of recombinant peptides from the GR containing AF1 have shown it to have little or no structure in simple buffer solutions (10). In the presence of the strong ␣-helix stabilizing agent trifluoroethanol, up to three ␣-helices could form at the C-terminal end of AF1, and functional mutagenesis has shown that the primary sequences at the C-terminal end of AF1 may...