Trimethylamine N-Oxide-induced Cooperative Folding of an Intrinsically Unfolded Transcription-activating Fragment of Human Glucocorticoid Receptor

Abstract

A number of biologically important proteins or protein domains identified recently are fully or partially unstructured (unfolded). Methods that allow studies of the propensity of such proteins to fold naturally are valuable. The traditional biophysical approaches using alcohols to drive ␣-helix formation raise serious questions of the relevance of alcohol-induced structure to the biologically important conformations. Recently we illustrated the extraordinary capability of the naturally occurring solute, trimethylamine N-oxide (TMAO), to force two unfolded proteins to fold to native-like species with significant functional activity. In the present work we apply this technique to recombinant human glucocorticoid receptor fragments consisting of residues 1-500 and residues 77-262. CD and fluorescence spectroscopy showed that both were largely disordered in aqueous solution. TMAO induced a condensed structure in the large fragment, indicated by the substantial enhancement in intrinsic fluorescence and blue shift of fluorescent maxima. CD spectroscopy demonstrated that the TMAO-induced structure is different from the ␣-helix-rich conformation driven by trifluoroethanol (TFE). In contrast to TFE, the conformational transition of the 1-500 fragment induced by TMAO is cooperative, a condition characteristic of proteins with unique structures.An increasing number of biologically important proteins and protein domains have been found to be only partially structured or unstructured (unfolded) under physiological conditions (1). Notably, many of the nuclear transcription factors show disordered transactivation domains in aqueous solution (2). It is generally accepted that the structural uniqueness of proteins determines their biological function. Hence, the identification of unstructured proteins raises the question: what is the structural basis of the functional activity of such proteins/ domains? Whether they act being in unfolded state ("natively unfolded" proteins) or adopt structure upon specific interaction with target molecules is a crucial question. The induced-fit and acidic blob models of the function for such transcription factor proteins represent two opposite points of view (3). Hence, methods that allow studies of the propensity of proteins to fold naturally are valuable.Alcohols (trifluoroethanol, chloroethanol) have long been used to probe the propensity of unstructured protein/domain to form secondary structure (4 -6). Their use has in part been based on the assumption that alcohols might mimic the in vivo conditions under which the disordered domain interacts with a target molecule. It has long been known, however, that alcohols favor the ␣-helical conformation in peptides or proteins regardless of the type of the secondary structure the proteins/peptides form in the biologically relevant (native) conformation (7-9). Hence, until interacting partners of unstructured domains are identified, the current biophysical approaches using such alcohols to drive ␣-helix formation present serious difficulties in inte...

TATA box binding protein induces structure in the recombinant glucocorticoid receptor AF1 domain

“…The primary amino acid sequences of the NTDs of the steroid receptors, which contain transactivation function region AF1, are much less conserved than are DBD and ligand binding domain regions, and several studies have shown that the isolated NTDs are not fully structured in aqueous solution (17,18,27,28). Although the isolated NTDs of steroid receptors have poor sequence homology, in solution they share the characteristic of disordered structure.…”

“…The GR AF1 sequence resembles those of acidic transactivation domains of several transcription factors (15,16). When expressed independently, the GR AF1 shows little structure and seems to exist as an ensemble of largely unstructured conformers (17,18). The GR AF1 is known to interact with other transcription factors, and conditional folding has been suggested to be the key for these interactions (19)(20)(21)(22).…”

An Androgen Receptor NH2-terminal Conserved Motif Interacts with the COOH Terminus of the Hsp70-interacting Protein (CHIP)

“…Analysis of the predicted order and disorder composition of the AR NH 2 -terminal domain using the PONDR Protein Disorder Predictor 3 indicates that the FXXLF motif and the AR NH 2 -terminal conserved motif form short structurally ordered regions as indicated by PONDR scores of Ͻ0.1. Each motif is predicted to form an amphipathic ␣-helix, indicating the presence of short structured segments in an NH 2 -terminal region that is otherwise predicted to be largely unstructured.…”

Binary Phases of Aliphatic N-Oxides and Water: Force Field Development and Molecular Dynamics Simulation

“…11 Explanations on the molecular level for these phenomena are only beginning to surface. [12][13][14][15][16][17][18][19] To investigate into the molecular mechanism of these effects by computational methods such as molecular dynamics (MD) simulation techniques, a force field for mixed solvents composed of water and N-oxide species is required that is also compatible with available biopolymer potential energy functions. Noto et al 20 were the first who constructed a force field for a rigid TMAO model in the presence of an aqueous environment based on quantum-chemical calculations of TMAO and a single water molecule within the Hartree-Fock (HF) approximation.…”