Guillaume Bouvignies

Solution structure of a minor and transiently formed state of a T4 lysozyme mutant

Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers). However, a rigorous understanding of the relation between a protein’s structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules and populates an excited state transiently (about 1 ms) to about 3% at 25 °C (ref. 5). We show that such binding occurs only via the ground state, and present the atomic-level model of the ‘invisible’, excited state obtained using a combined strategy of relaxation-dispersion NMR (ref. 6) and CS-Rosetta model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein’s function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states.

Protein Conformational Flexibility from Structure-Free Analysis of NMR Dipolar Couplings: Quantitative and Absolute Determination of Backbone Motion in Ubiquitin†

A robust procedure for the determination of protein-backbone motions on time scales of pico- to milliseconds directly from residual dipolar couplings has been developed that requires no additional scaling relative to external references. The results for ubiquitin (blue in graph: experimental N-HN order parameters) correspond closely to the amplitude, nature, and distribution of motion found in a 400 ns molecular-dynamics trajectory of ubiquitin (red).

13CHD2 methyl group probes of millisecond time scale exchange in proteins by 1H relaxation dispersion: an application to proteasome gating residue dynamics.

A pulse scheme is presented for quantifying millisecond time scale chemical exchange processes in proteins by measuring (1)H CPMG relaxation dispersion profiles of (13)CHD(2) methyl groups. The use of (13)CHD(2) isotopomers for (1)H methyl dispersion experiments eliminates problems with interconversion between differentially relaxing proton transitions that complicate the extraction of accurate exchange parameters when (13)CH(3) probes are used. Good agreement is demonstrated between extracted chemical shift differences from fits of dispersion profiles and the corresponding differences measured independently on a model exchanging system, validating the experiment. The methodology is applied to the gating residues of the T. acidiphilium proteasome that are shown to undergo extensive motion on the millisecond time scale.

Nuclear magnetic resonance provides a quantitative description of protein conformational flexibility on physiologically important time scales.

A complete description of biomolecular activity requires an understanding of the nature and the role of protein conformational dynamics. In recent years, novel nuclear magnetic resonance-based techniques that provide hitherto inaccessible detail concerning biomolecular motions occurring on physiologically important time scales have emerged. Residual dipolar couplings (RDCs) provide precise information about time- and ensemble-averaged structural and dynamic processes with correlation times up to the millisecond and thereby encode key information for understanding biological activity. In this review, we present the application of two very different approaches to the quantitative description of protein motion using RDCs. The first is purely analytical, describing backbone dynamics in terms of diffusive motions of each peptide plane, using extensive statistical analysis to validate the proposed dynamic modes. The second is based on restraint-free accelerated molecular dynamics simulation, providing statistically sampled free energy-weighted ensembles that describe conformational fluctuations occurring on time scales from pico- to milliseconds, at atomic resolution. Remarkably, the results from these two approaches converge closely in terms of distribution and absolute amplitude of motions, suggesting that this kind of combination of analytical and numerical models is now capable of providing a unified description of protein conformational dynamics in solution.

Measurement of proton chemical shifts in invisible states of slowly exchanging protein systems by chemical exchange saturation transfer.

Chemical exchange saturation transfer (CEST) NMR spectroscopy has emerged as a powerful technique for studies of transiently formed, sparsely populated (excited) conformational states of protein molecules in slow exchange with a dominant structure. The most popular form of the experiment, and the version originally developed, uses a weak (1)H radio frequency field to perturb longitudinal magnetization of one state with the effect transferred to magnetization in the second conformation via chemical exchange. A significant limitation of the method for protein applications emerges from (1)H magnetization transfer via dipolar relaxation (NOE effect) that can severely complicate analysis of the resulting CEST profile. This is particularly an issue since the (1)H chemical shifts of the excited state, critical for structural studies of these elusive conformers, become difficult to extract. Here we present a method for measurement of these shifts via CEST experiments in which the NOE effect is not an issue. The methodology is illustrated through applications to a pair of exchanging systems where the results are cross-validated.

Thermal fluctuations of immature SOD1 lead to separate folding and misfolding pathways

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease involving cytotoxic conformations of Cu, Zn superoxide dismutase (SOD1). A major challenge in understanding ALS disease pathology has been the identification and atomic-level characterization of these conformers. Here, we use a combination of NMR methods to detect four distinct sparsely populated and transiently formed thermally accessible conformers in equilibrium with the native state of immature SOD1 (apoSOD12SH). Structural models of two of these establish that they possess features present in the mature dimeric protein. In contrast, the other two are non-native oligomers in which the native dimer interface and the electrostatic loop mediate the formation of aberrant intermolecular interactions. Our results show that apoSOD12SH has a rugged free energy landscape that codes for distinct kinetic pathways leading to either maturation or non-native association and provide a starting point for a detailed atomic-level understanding of the mechanisms of SOD1 oligomerization. DOI: http://dx.doi.org/10.7554/eLife.07296.001

A 2D 13C-CEST experiment for studying slowly exchanging protein systems using methyl probes: an application to protein folding

A 2D 13C Chemical Exchange Saturation Transfer (CEST) experiment is presented for studying slowly exchanging protein systems using methyl groups as probes. The utility of the method is first established through studies of protein L, a small protein, for which chemical exchange on the millisecond time-scale is not observed. Subsequently the approach is applied to a folding exchange reaction of a G48M mutant Fyn SH3 domain, for which only cross-peaks derived from the folded (‘ground’) state are present in spectra. Fits of 15N and methyl 13C CEST profiles of the Fyn SH3 domain establish that the exchange reaction involves an interchange between folded and unfolded conformers, although elevated methyl 13C transverse relaxation rates for some of the residues of the unfolded (‘invisible, excited’) state indicate that it likely exchanges with a third conformation as well. In addition to the kinetics of the exchange reaction, methyl carbon chemical shifts of the excited state are also obtained from analysis of the 13C CEST data.

16-Fold Degeneracy of Peptide Plane Orientations from Residual Dipolar Couplings: Analytical Treatment and Implications for Protein Structure Determination

Residual dipolar couplings (RDCs) measured for internally rigid molecular fragments provide important information about the relative orientations of these fragments. Dependent on the symmetry of the alignment tensor and the symmetry of the molecular fragment, however, there generally exist more than one solution for the fragment orientation consistent with the measured RDCs. Analytical solutions are presented that describe the complete set of orientations of internally rigid fragments that are consistent with multiple dipolar couplings measured in a single alignment medium that is rhombic. For the first time, it is shown that, for a planar fragment such as the peptide plane, there generally exist 16 different solutions with their analytical expressions presented explicitly. The presence of these solutions is shown to be highly relevant for standard structure determination protocols using RDCs to refine molecular structures. In particular, when using standard protein structure refinement with RDCs that were measured in a single alignment medium as constraints, it is found that often more than one of the peptide plane solutions is physically viable; i.e., despite being consistent with measured RDCs, the local backbone structure can be incorrect. On the basis of experimental and simulated examples, it is rationalized why protein structures that are refined against RDCs measured in a single medium can have lower resolution (precision) than one would expect on the basis of the experimental accuracy of the RDCs. Conditions are discussed under which the correct solution can be identified.

Mapping the conformation of a client protein through the Hsp70 functional cycle

Significance Hsp70 chaperones are key components of the cellular proteostasis network. The ATP-dependent interaction of Hsp70 with its substrates prevents aggregation and promotes their correct folding and maturation. Here we investigate the impact of Hsp70 binding on the conformation of a client substrate using nuclear magnetic resonance spectroscopy. Our experiments provide a model for Hsp70 action in which the substrate can adopt substantial amounts of secondary structure even in the globally unfolded Hsp70-bound state. Moreover, the substrate conformation does not evolve as a function of the Hsp70 nucleotide state, demonstrating that the large structural changes in Hsp70 resulting from ATP binding and hydrolysis do not perform conformational work on the bound substrate or lead to changes in client protein conformation. The 70 kDa heat shock protein (Hsp70) chaperone system is ubiquitous, highly conserved, and involved in a myriad of diverse cellular processes. Its function relies on nucleotide-dependent interactions with client proteins, yet the structural features of folding-competent substrates in their Hsp70-bound state remain poorly understood. Here we use NMR spectroscopy to study the human telomere repeat binding factor 1 (hTRF1) in complex with Escherichia coli Hsp70 (DnaK). In the complex, hTRF1 is globally unfolded with up to 40% helical secondary structure in regions distal to the binding site. Very similar conformational ensembles are observed for hTRF1 bound to ATP-, ADP- and nucleotide-free DnaK. The patterns in substrate helicity mirror those found in the unfolded state in the absence of denaturants except near the site of chaperone binding, demonstrating that DnaK-bound hTRF1 retains its intrinsic structural preferences. To our knowledge, our study presents the first atomic resolution structural characterization of a client protein bound to each of the three nucleotide states of DnaK and establishes that the large structural changes in DnaK and the associated energy that accompanies ATP binding and hydrolysis do not affect the overall conformation of the bound substrate protein.

Visualizing Side Chains of Invisible Protein Conformers by Solution NMR

Sparsely populated and transiently formed protein conformers can play key roles in many biochemical processes. Understanding the structure function paradigm requires, therefore, an atomic-resolution description of these rare states. However, they are difficult to study because they cannot be observed using standard biophysical techniques. In the past decade, NMR methods have been developed for structural studies of these elusive conformers, focusing primarily on backbone (1)H, (15)N and (13)C nuclei. Here we extend the methodology to include side chains by developing a (13)C-based chemical exchange saturation transfer experiment for the assignment of side-chain aliphatic (13)C chemical shifts in uniformly (13)C labeled proteins. A pair of applications is provided, involving the folding of β-sheet Fyn SH3 and α-helical FF domains. Over 96% and 89% of the side-chain (13)C chemical shifts for excited states corresponding to the unfolded conformation of the Fyn SH3 domain and a folding intermediate of the FF domain, respectively, have been obtained, providing insight into side-chain packing and dynamics.