Jie-rong Huang

Exploring Free-Energy Landscapes of Intrinsically Disordered Proteins at Atomic Resolution Using NMR Spectroscopy

Proteins at Atomic Resolution Using NMR Spectroscopy Malene Ringkjøbing Jensen,†,‡,§ Markus Zweckstetter, Jie-rong Huang,†,‡,§ and Martin Blackledge*,†,‡,§ †Universite ́ Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38027 Grenoble, France ‡CEA, DSV, IBS, F-38027 Grenoble, France CNRS, IBS, F-38027 Grenoble, France Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany German Center for Neurodegenerative Diseases (DZNE), 37077 Göttingen, Germany Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), University Medical Center, 37073 Göttingen, Germany

DOTA-M8: An extremely rigid, high-affinity lanthanide chelating tag for PCS NMR spectroscopy.

A new lanthanide chelating tag (M8) for paramagnetic labeling of biomolecules is presented, which is based on an eight-fold, stereoselectively methyl-substituted DOTA that can be covalently linked to the host molecule by a single disulfide bond. The steric overcrowding of the DOTA scaffold leads to an extremely rigid, kinetically and chemically inert lanthanide chelator. Its steric bulk restricts the motion of the tag relative to the host molecule. These properties result in very large pseudocontact shifts (>5 ppm) and residual dipolar couplings (>20 Hz) for Dy-M8 linked to ubiquitin, which are unprecedented for a small, single-point-attachment tag. Such large pseudocontact shifts should be well detectable even for larger proteins and distances beyond approximately 50 A. Due to its exceptionally high stability and lanthanide affinity M8 can be used under extreme chemical or physical conditions, such as those applied for protein denaturation, or when it is undesirable that buffer or protein react with excess lanthanide ions.

Ensemble calculations of unstructured proteins constrained by RDC and PRE data: a case study of urea-denatured ubiquitin.

The detailed, quantitative characterization of unfolded proteins is a largely unresolved task due to the enormous experimental and theoretical difficulties in describing the highly dimensional space of their conformational ensembles. Recently, residual dipolar coupling (RDC) and paramagnetic relaxation enhancement (PRE) data have provided large numbers of experimental parameters on unfolded states. To obtain a minimal model of the unfolded state according to such data we have developed new modules for the use of steric alignment RDCs and PREs as constraints in ensemble structure calculations by the program XPLOR-NIH. As an example, ensemble calculations were carried out on urea-denatured ubiquitin using a total of 419 previously obtained RDCs and 253 newly determined PREs from eight cysteine mutants coupled to MTSL. The results show that only a small number of about 10 conformers is necessary to fully reproduce the experimental RDCs, PREs and average radius of gyration. C(alpha) contacts determined on a large set (400) of 10-conformer ensembles show significant (10-20%) populations of conformations that are similar to ubiquitins A-state, i.e. corresponding to an intact native first beta-hairpin and alpha-helix as well as non-native alpha-helical conformations in the C-terminal half. Thus, methanol/acid (A-state) and urea denaturation lead to similar low energy states of the protein ensemble, presumably due to the weakening of the hydrophobic core. Similar contacts are obtained in calculations using solely RDCs or PREs. The sampling statistics of the C(alpha) contacts in the ensembles follow a simple binomial distribution. It follows that the present RDC, PRE, and computational methods allow the statistically significant detection of subconformations in the unfolded ensemble at population levels of a few percent.

Mapping the Potential Energy Landscape of Intrinsically Disordered Proteins at Amino Acid Resolution

Intrinsically disordered regions are predicted to exist in a significant fraction of proteins encoded in eukaryotic genomes. The high levels of conformational plasticity of this class of proteins endows them with unique capacities to act in functional modes not achievable by folded proteins, but also places their molecular characterization beyond the reach of classical structural biology. New techniques are therefore required to understand the relationship between primary sequence and biological function in this class of proteins. Although dependences of some NMR parameters such as chemical shifts (CSs) or residual dipolar couplings (RDCs) on structural propensity are known, so that sampling regimes are often inferred from experimental observation, there is currently no framework that allows for a statistical mapping of the available Ramachandran space of each amino acid in terms of conformational propensity. In this study we develop such an approach, combining highly efficient conformational sampling with ensemble selection to map the backbone conformational sampling of IDPs on a residue specific level. By systematically analyzing the ability of NMR data to map the conformational landscape of disordered proteins, we identify combinations of RDCs and CSs that can be used to raise conformational degeneracies inherent to different data types, and apply these approaches to characterize the conformational behavior of two intrinsically disordered proteins, the K18 domain from Tau protein and N(TAIL) from measles virus nucleoprotein. In both cases, we identify the enhanced populations of turn and helical regions in key regions of the proteins, as well as contiguous strands that show clear and enhanced polyproline II sampling.

Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS.

Significance Proteins are the most versatile components of the molecular machinery of life. Synthesized as linear polymers of amino acids, proteins start out in their unfolded state and perform their function either in well-defined folded conformations or as intrinsically disordered proteins (IDPs) lacking tertiary structure. Both for the folding process and the properties of IDPs, a quantitative understanding of the conformational distributions and dynamics of unfolded proteins is thus essential. However, reaching this goal has been challenging owing to the large conformational heterogeneity and rapid dynamics of these systems. Here we combine three of the most powerful biophysical methods available to obtain a comprehensive view of an unfolded protein that would not be available from any of the individual methods. The properties of unfolded proteins are essential both for the mechanisms of protein folding and for the function of the large group of intrinsically disordered proteins. However, the detailed structural and dynamical characterization of these highly dynamic and conformationally heterogeneous ensembles has remained challenging. Here we combine and compare three of the leading techniques for the investigation of unfolded proteins, NMR spectroscopy (NMR), small-angle X-ray scattering (SAXS), and single-molecule Förster resonance energy transfer (FRET), with the goal of quantitatively testing their consistency and complementarity and for obtaining a comprehensive view of the unfolded-state ensemble. Using unfolded ubiquitin as a test case, we find that its average dimensions derived from FRET and from structural ensembles calculated using the program X-PLOR-NIH based on NMR and SAXS restraints agree remarkably well; even the shapes of the underlying intramolecular distance distributions are in good agreement, attesting to the reliability of the approaches. The NMR-based results provide a highly sensitive way of quantifying residual structure in the unfolded state. FRET-based nanosecond fluorescence correlation spectroscopy allows long-range distances and chain dynamics to be probed in a time range inaccessible by NMR. The combined techniques thus provide a way of optimally using the complementarity of the available methods for a quantitative structural and dynamical description of unfolded proteins both at the global and the local level.

Understanding the folding of GFP using biophysical techniques

Green fluorescent protein (GFP) and its many variants are probably the most widely used proteins in medical and biological research, having been extensively engineered to act as markers of gene expression and protein localization, indicators of protein–protein interactions and biosensors. GFP first folds, before it can undergo an autocatalytic cyclization and oxidation reaction to form the chromophore, and in many applications the folding efficiency of GFP is known to limit its use. Here, we review the recent literature on protein engineering studies that have improved the folding properties of GFP. In addition, we discuss in detail the biophysical work on the folding of GFP that is beginning to reveal how this large and complex structure forms.

Transient electrostatic interactions dominate the conformational equilibrium sampled by multidomain splicing factor U2AF65: a combined NMR and SAXS study.

Multidomain proteins containing intrinsically disordered linkers exhibit large-scale dynamic modes that play key roles in a multitude of molecular recognition and signaling processes. Here, we determine the conformational space sampled by the multidomain splicing factor U2AF65 using complementary nuclear magnetic resonance spectroscopy and small-angle scattering data. Available degrees of conformational freedom are initially stochastically sampled and experimental data then used to delineate the potential energy landscape in terms of statistical probability. The spatial distribution of U2AF65 conformations is found to be highly anisotropic, comprising significantly populated interdomain contacts that appear to be electrostatic in origin. This hypothesis is supported by the reduction of signature PREs reporting on expected interfaces with increasing salt concentration. The described spatial distribution reveals the complete spectrum of the unbound forms of U2AF65 that coexist with the small percentage of a preformed RNA-bound domain arrangement required for polypyrimidine-tract recognition by conformational selection. More generally, the proposed approach to describing conformational equilibria of multidomain proteins can be further combined with other experimental data that are sensitive to domain dynamics.

Sequence-Specific Mapping of the Interaction between Urea and Unfolded Ubiquitin from Ensemble Analysis of NMR and Small Angle Scattering Data

The molecular details of how urea interacts with, and eventually denatures proteins, remain largely unknown. In this study we have used extensive experimental NMR data, in combination with statistical coil ensemble modeling and small-angle scattering, to analyze the conformational behavior of the protein ubiquitin in the presence of urea. In order to develop an atomic resolution understanding of the denatured state, conformational ensembles of full-atom descriptions of unfolded proteins, including side chain conformations derived from rotamer libraries, are combined with random sampling of explicit urea molecules in interaction with the protein. Using this description of the conformational equilibrium, we demonstrate that the direct-binding model of urea to the protein backbone is compatible with available experimental data. We find that, in the presence of 8 M urea, between 30 and 40% of the backbone peptide groups bind a urea molecule, independently reproducing results from a model-free analysis of small-angle neutron and X-ray scattering data. Crucially, this analysis also provides sequence specific details of the interaction between urea and the protein backbone. The pattern of urea-binding along the amino-acid sequence reveals a higher level of binding in the central part of the protein, a trend which resembles independent results derived from chemical shift mapping of the urea-protein interaction. Together these results substantiate the direct-binding model and provide a framework for studying the physical basis of interactions between proteins and solvent molecules.

Modulation of structure and dynamics by disulfide bond formation in unfolded states.

During oxidative folding, the formation of disulfide bonds has profound effects on guiding the protein folding pathway. Until now, comparatively little is known about the changes in the conformational dynamics in folding intermediates of proteins that contain only a subset of their native disulfide bonds. In this comprehensive study, we probe the conformational landscape of non-native states of lysozyme containing a single native disulfide bond utilizing nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), circular dichroism (CD) data, and modeling approaches. The impact on conformational dynamics varies widely depending on the loop size of the single disulfide variants and deviates significantly from random coil predictions for both NMR and SAXS data. From these experiments, we conclude that the introduction of single disulfides spanning a large portion of the polypeptide chain shifts the structure and dynamics of hydrophobic core residues of the protein so that these regions exhibit levels of order comparable to the native state on the nanosecond time scale.

The extremely slow-exchanging core and acid-denatured state of green fluorescent protein

Green fluorescent protein (GFP) is a large protein with a complex eleven‐stranded β‐barrel structure. Previous studies have shown that it has a complex energy landscape for folding on which there are several intermediate states and a denatured state with significant residual structure. Here, we use two different types of H/D exchange measurement and nuclear magnetic resonance (NMR) techniques to probe the energy landscape for folding of GFP in further detail. H/D exchange experiments were performed over a wide range of conditions including different concentrations of denaturant. Results show that the penetration model dominates the exchange mechanism, consistent with the known stability and slow unfolding kinetics of GFP. H/D exchange experiments at high pH establish that there is an extremely slow‐exchanging superstable core of amide protons in GFP that are clustered and located in β‐strands 1, 2, 4, 5, and 6. These residues form part of a mini‐β‐sheet which we propose constitutes a folding nucleus. Using a pulsed‐labeling strategy, the acid‐denatured state has been investigated and the residual structure observed in earlier studies shown to locate to β‐strands 1 and 3. There is some evidence that this residual structure is stabilized by a localized hydrophobic collapse of the polypeptide chain.