Native molten globules are the most folded kind of intrinsically disordered proteins. Little is known about the mechanism by which native molten globules bind to their cognate ligands to form fully folded complexes. The nuclear coactivator binding domain (NCBD) of CREB binding protein is particularly interesting in this respect as structural studies of its complexes have shown that NCBD folds into two remarkably different states depending on the ligand being ACTR or IRF-3. The ligand-free state of NCBD was characterized in order to understand the mechanism of folding upon ligand binding. Biophysical studies show that despite the molten globule nature of the domain, it contains a small cooperatively folded core. By NMR spectroscopy, we have demonstrated that the folded core of NCBD has a well ordered conformer with specific side chain packing. This conformer resembles the structure of the NCBD in complex with the protein ligand, ACTR, suggesting that ACTR binds to prefolded NCBD molecules from the ensemble of interconverting structures.CREB binding protein | folding upon binding | NMR spectroscopy T he protein structure-function paradigm has dominated structural and molecular biology over the past five decades. This paradigm has been challenged by the discovery of functional but intrinsically disordered proteins (IDPs) (1). IDPs are abundant in higher organisms and are necessary for many crucial biological functions such as cell cycle regulation, signal transduction, and regulation of transcription (2, 3). Structural studies of IDPs have shown that despite the high degree of disorder, these proteins are far from randomly structured and form transient, yet specific, secondary and tertiary structural elements (4, 5). The success of the structure-function paradigm in explaining the function of folded proteins suggests that the functions of the IDPs may also be understood by studying the transient structure of the disordered state.The molten globule was originally discovered as a partially folded state under mildly denaturing conditions and was shown to accumulate as an intermediate during protein folding reactions (6, 7). With the emergence of the IDPs, proteins were discovered where the molten globule is the functionally active state. Native molten globules are the most compact conformational state considered as IDPs (4). Molten globules have native-like secondary structure, but are believed to lack the well-defined tertiary interactions found in folded proteins. Most high resolution NMR studies of the molten globule state have focused on the secondary structure of the backbone as this is more readily accessible from secondary chemical shifts (8). The degree of side chain packing, however, is less well understood. Molten globules are believed to have dynamic hydrophobic cores due to the lack of signal in the near-UV CD spectrum and the broadening of NMR signals of aromatic groups (6, 7). Recent studies have shown that the side chains in the α-lactalbumin molten globule exchange between several well defined confor...

The denatured state of a protein contains important information about the determinants of the folding process. By combining site-directed spin-labeling NMR experiments and restrained computer simulations, we have determined ensembles of conformations that represent the denatured state of the bovine acyl-coenzyme A binding protein (ACBP) at three different concentrations of guanidine hydrochloride. As the experimentally determined distance information corresponds to weighted averages over a broad ensemble of structures, we applied the experimental restraints to a system of noninteracting replicas of the protein by using a Monte Carlo sampling scheme. This procedure permits us to sample ensembles of conformations that are compatible with the experimental data and thus to obtain information regarding the distribution of structures in the denatured state. Our results show that the denatured state of ACBP is highly heterogeneous. The high sensitivity of the computational method that we present, however, enabled us to identify long-range interactions between two regions, located near the N- and C-termini, that include both native and non-native elements. The preferential formation of these contacts suggests that the sequence-dependent patterns of helical propensity and hydrophobicity are important determinants of the structure in the denatured state of ACBP.

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a Reprint requests to: Kevin W. Plaxco, Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA; e-mail: kwp@chem.ucsb.edu; fax: (805) 893-4120.Abbreviations: GuHCl, guanidine hydrochloride; tris, tris hydroxymethylaminoethane; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TCEP, tris(2-carboxyethyl)phosphine; CD, circular dichroism. Article published online ahead of print. Article and publication date are at

A simple labeling approach is presented based on protein expression in [1-(13)C]- or [2-(13)C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C(alpha) sites, respectively. All of the methyl groups, with the exception of Thr and Ile(delta1) are produced with isolated (13)C spins (i.e., no (13)C-(13)C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-alpha sites are labeled without concomitant labeling at C(beta) positions for 17 of the common 20 amino acids and there are no cases for which (13)C(alpha)-(13)CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone (15)N studies. The utility of the labeling is established by recording (13)C R (1rho) and CPMG-based experiments on a number of different protein systems.