A pre-existing hydrophobic collapse in the unfolded state of an ultrafast folding protein

Abstract

Insights into the conformational passage of a polypeptide chain across its free energy landscape have come from the judicious combination of experimental studies and computer simulations 1,2 . Even though some unfolded and partially folded proteins are now known to possess biological function 3 or to be involved in aggregation phenomena associated with disease states 1,4 , experimentally derived atomic-level information on these structures remains sparse as a result of conformational heterogeneity and dynamics. Here we present a technique that can provide such information. Using a 'Trp-cage' miniprotein known as TC5b (ref. 5), we report photochemically induced dynamic nuclear polarization NMR 6 pulse-labelling experiments that involve rapid in situ protein refolding 7,8 . These experiments allow dipolar cross-relaxation with hyperpolarized aromatic side chain nuclei in the unfolded state to be identified and quantified in the resulting folded-state spectrum. We find that there is residual structure due to hydrophobic collapse in the unfolded state of this small protein, with strong inter-residue contacts between side chains that are relatively distant from one another in the native state. Prior structuring, even with the formation of non-native rather than native contacts, may be a feature associated with fast folding events in proteins.Experimental advances in nuclear magnetic resonance (NMR) spectroscopy have led to the characterization of a diverse range of unfolded states of proteins 9 . In many cases the presence of residual structure has been shown 10-13 , but with some significant exceptions 14 the poorly resolved spectra of the unfolded state, arising from conformational exchange and dynamic averaging, have generally hampered structural analysis by NMR. We report here the use of an NMR technique that circumvents some of these problems by transferring Three methodologies are combined in this 'pulse-labelling' experiment ( Fig. 1). (1) Photo-CIDNP (chemically induced dynamic nuclear polarization) 6,22 , a technique for enhancing the NMR signals ('hyperpolarization') of solvent-accessible tryptophan, tyrosine and histidine side chains by means of a laser-induced reaction of the protein with a flavin photosensitizer. (2) Rapid homogeneous mixing of solutions in the NMR sample tube to trigger the folding of a denatured protein on a timescale faster than nuclear spin-lattice relaxation ( Supplementary Fig. 1) 7,8 . To these two techniques we add here, for the first time, (3) transfer of nuclear magnetization via nuclear Overhauser effects (NOEs) from the hyperpolarized side chain protons to neighbouring atoms before the refolding step. As a result, inter-residue contacts in unfolded conformations can be detected in the well-resolved NMR spectrum of the refolded native state.1 H photo-CIDNP measurements were initially performed on the native and denatured states of TC5b. The photo-CIDNP spectrum of native TC5b (Fig. 2b) is considerably simpler than the conventional NMR spectrum (Fig. 2a), because only t...

Measurement of energy landscape roughness of folded and unfolded proteins

“…Close inspection of triplet-quenching experiments on denatured proteins shows slight indications of nonexponential dynamics on the 10-100-μs timescale that might indicate deeper traps (18). The existence of significant interactions in unfolded proteins had been suggested previously on the basis of photochemically induced dynamic nuclear polarization NOE (49) and paramagnetic relaxation enhancement (50) NMR experiments and from the low value and unusual denaturant dependence of their intrapolymer diffusion constant (18). Similarly, the radius of gyration of the unfolded state of staphylococcal nuclease indicated the formation of transient hydrophobic clusters (51).…”

Crystal and NMR structures of a Trp-cage mini-protein benchmark for computational fold prediction

“…Trp-cage species have been extensively studied experimentally (4)(5)(6)(7)(8)(9)(10)(11) and computationally (12)(13)(14)(15)(16)(17)(18)(19), but the conclusions regarding the folding process and results drawn from these studies are not in accord at the level that should be attainable for such a small system.…”

“…protein-protein interaction | folding simulation target | protein cyclization | peptide oligomerization | H-bonding motifs T rp-cage miniproteins (at 18-20 residue length) are among the smallest peptides to fold into a stable protein-like structure (1,2); as a result, the Trp-cage fold has emerged as a protein folding paradigm (3,4). Trp-cage species have been extensively studied experimentally (4)(5)(6)(7)(8)(9)(10)(11) and computationally (12)(13)(14)(15)(16)(17)(18)(19), but the conclusions regarding the folding process and results drawn from these studies are not in accord at the level that should be attainable for such a small system.…”

Energetically significant networks of coupled interactions within an unfolded protein

“…These are a critical piece of the folding puzzle; they impact protein aggregation and folding, and have important implications for the properties of IDPs. Recent studies offer evidence for hydrophobic clusters, electrostatic interactions, and residual secondary structure in the DSE under near-native conditions (20)(21)(22). This naturally leads to the questions of how DSE interactions contribute to protein stability and folding, and if networks of coupled interactions occur in the DSE.…”