Xavier Salvatella

Protein structure determination from NMR chemical shifts

NMR spectroscopy plays a major role in the determination of the structures and dynamics of proteins and other biological macromolecules. Chemical shifts are the most readily and accurately measurable NMR parameters, and they reflect with great specificity the conformations of native and nonnative states of proteins. We show, using 11 examples of proteins representative of the major structural classes and containing up to 123 residues, that it is possible to use chemical shifts as structural restraints in combination with a conventional molecular mechanics force field to determine the conformations of proteins at a resolution of 2 Å or better. This strategy should be widely applicable and, subject to further development, will enable quantitative structural analysis to be carried out to address a range of complex biological problems not accessible to current structural techniques.

Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR

Many biochemical processes proceed through the formation of functionally significant intermediates. Although the identification and characterization of such species can provide vital clues about the mechanisms of the reactions involved, it is challenging to obtain information of this type in cases where the intermediates are transient or present only at low population. One important example of such a situation involves the folding behaviour of small proteins that represents a model for the acquisition of functional structure in biology. Here we use relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy to identify, for two mutational variants of one such protein, the SH3 domain from Fyn tyrosine kinase, a low-population folding intermediate in equilibrium with its unfolded and fully folded states. By performing the NMR experiments at different temperatures, this approach has enabled characterization of the kinetics and energetics of the folding process as well as providing structures of the intermediates. A general strategy emerges for an experimental determination of the energy landscape of a protein by applying this methodology to a series of mutants whose intermediates have differing degrees of native-like structure.

Fast and accurate predictions of protein NMR chemical shifts from interatomic distances.

We present a method, CamShift, for the rapid and accurate prediction of NMR chemical shifts from protein structures. The calculations performed by CamShift are based on an approximate expression of the chemical shifts in terms of polynomial functions of interatomic distances. Since these functions are very fast to compute and readily differentiable, the CamShift approach can be utilized in standard protein structure calculation protocols.

Weak Long-Range Correlated Motions in a Surface Patch of Ubiquitin Involved in Molecular Recognition

Long-range correlated motions in proteins are candidate mechanisms for processes that require information transfer across protein structures, such as allostery and signal transduction. However, the observation of backbone correlations between distant residues has remained elusive, and only local correlations have been revealed using residual dipolar couplings measured by NMR spectroscopy. In this work, we experimentally identified and characterized collective motions spanning four β-strands separated by up to 15 Å in ubiquitin. The observed correlations link molecular recognition sites and result from concerted conformational changes that are in part mediated by the hydrogen-bonding network.

Peptide nanofibrils boost retroviral gene transfer and provide a rapid means for concentrating viruses

Inefficient gene transfer and low virion concentrations are common limitations of retroviral transduction. We and others have previously shown that peptides derived from human semen form amyloid fibrils that boost retroviral gene delivery by promoting virion attachment to the target cells. However, application of these natural fibril-forming peptides is limited by moderate efficiencies, the high costs of peptide synthesis, and variability in fibril size and formation kinetics. Here, we report the development of nanofibrils that self-assemble in aqueous solution from a 12-residue peptide, termed enhancing factor C (EF-C). These artificial nanofibrils enhance retroviral gene transfer substantially more efficiently than semen-derived fibrils or other transduction enhancers. Moreover, EF-C nanofibrils allow the concentration of retroviral vectors by conventional low-speed centrifugation, and are safe and effective, as assessed in an ex vivo gene transfer study. Our results show that EF-C fibrils comprise a highly versatile, convenient and broadly applicable nanomaterial that holds the potential to significantly facilitate retroviral gene transfer in basic research and clinical applications.

The Non-Core Regions of Human Lysozyme Amyloid Fibrils Influence Cytotoxicity

Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of aggregates formed by lysozyme, a protein found as fibrillar deposits in patients with familial systemic amyloidosis, by infrared spectroscopy, transmission electron microscopy, and depolymerization experiments, and analyzed how they affect cell viability. We have characterized two types of human lysozyme amyloid structures formed in vitro that differ in morphology, molecular structure, stability, and size of the cross-β core. Of particular interest is that the fibrils with a smaller core generate a significant cytotoxic effect. These findings indicate that protein aggregation can give rise to species with different degree of cytotoxicity due to intrinsic differences in their physicochemical properties.

Understanding biomolecular motion, recognition, and allostery by use of conformational ensembles

We review the role conformational ensembles can play in the analysis of biomolecular dynamics, molecular recognition, and allostery. We introduce currently available methods for generating ensembles of biomolecules and illustrate their application with relevant examples from the literature. We show how, for binding, conformational ensembles provide a way of distinguishing the competing models of induced fit and conformational selection. For allostery we review the classic models and show how conformational ensembles can play a role in unravelling the intricate pathways of communication that enable allostery to occur. Finally, we discuss the limitations of conformational ensembles and highlight some potential applications for the future.

Time Averaging of NMR Chemical Shifts in the MLF Peptide in the Solid State

Since experimental measurements of NMR chemical shifts provide time and ensemble averaged values, we investigated how these effects should be included when chemical shifts are computed using density functional theory (DFT). We measured the chemical shifts of the N-formyl-L-methionyl-L-leucyl-L-phenylalanine-OMe (MLF) peptide in the solid state, and then used the X-ray structure to calculate the (13)C chemical shifts using the gauge including projector augmented wave (GIPAW) method, which accounts for the periodic nature of the crystal structure, obtaining an overall accuracy of 4.2 ppm. In order to understand the origin of the difference between experimental and calculated chemical shifts, we carried out first-principles molecular dynamics simulations to characterize the molecular motion of the MLF peptide on the picosecond time scale. We found that (13)C chemical shifts experience very rapid fluctuations of more than 20 ppm that are averaged out over less than 200 fs. Taking account of these fluctuations in the calculation of the chemical shifts resulted in an accuracy of 3.3 ppm. To investigate the effects of averaging over longer time scales we sampled the rotameric states populated by the MLF peptides in the solid state by performing a total of 5 micros classical molecular dynamics simulations. By averaging the chemical shifts over these rotameric states, we increased the accuracy of the chemical shift calculations to 3.0 ppm, with less than 1 ppm error in 10 out of 22 cases. These results suggests that better DFT-based predictions of chemical shifts of peptides and proteins will be achieved by developing improved computational strategies capable of taking into account the averaging process up to the millisecond time scale on which the chemical shift measurements report.

Population of Nonnative States of Lysozyme Variants Drives Amyloid Fibril Formation

The propensity of protein molecules to self-assemble into highly ordered, fibrillar aggregates lies at the heart of understanding many disorders ranging from Alzheimers disease to systemic lysozyme amyloidosis. In this paper we use highly accurate kinetic measurements of amyloid fibril growth in combination with spectroscopic tools to quantify the effect of modifications in solution conditions and in the amino acid sequence of human lysozyme on its propensity to form amyloid fibrils under acidic conditions. We elucidate and quantify the correlation between the rate of amyloid growth and the population of nonnative states, and we show that changes in amyloidogenicity are almost entirely due to alterations in the stability of the native state, while other regions of the global free-energy surface remain largely unmodified. These results provide insight into the complex dynamics of a macromolecule on a multidimensional energy landscape and point the way for a better understanding of amyloid diseases.

Kinetics of Conformational Sampling in Ubiquitin

Molecular recognition plays a central role in many biological processes. For enzymatic reactions and slow protein–protein recognition events, turn-over rates and on-rates in the millisecond-to-second time scale have been connected to internal protein dynamics detected with atomic resolution by NMR spectroscopy, and in particular conformational sampling could be established as a mechanism for enzyme–substrate and protein–protein recognition. Recent theoretical studies indicate that faster rates of conformational interconversion in the microsecond time scale might limit on-rates for protein–protein recognition. However experimental proofs were lacking so far, mainly because such rates could not be determined accurately enough and kinetic experiments in the microsecond time range are difficult to perform. Nevertheless, for proteins and TAR-RNA, recent studies based on residual dipolar couplings (RDCs) and other NMR spectroscopy techniques have detected substantial internal dynamics in a time window from the rotational correlation time tc (one-digit nanoseconds) to approximately 50 ms, called the supra-tc window in the following. However, the exact rates of internal dynamics within this four orders of magnitude wide time window could not be determined. Supra-tc dynamics in ubiquitin [9] and TAR-RNA could be connected to the conformational sampling required for molecular recognition. While the amplitudes of motions have been indirectly detected by RDCs and characterized in great detail, it has so far been impossible to directly observe these motions and to determine the exact rate of these supra-tc motions. In contrast, conformational sampling in enzymes occurs on a time scale that is 100 to 1000 times slower than supra-tc dynamics and therefore NMR relaxation dispersion (RD) techniques have been able to establish the functional link to enzyme kinetics with atomic resolution at physiological conditions. 5] However, for technical reasons, RD is not sensitive to motion faster than approximately 50 ms (RD window) and therefore does not access motion in the supra-tc window at room temperature. Here we determine the rate of interconversion between conformers of free ubiquitin by a combination of NMR RD experiments in super-cooled solution and dielectric relaxation spectroscopy (DR). Furthermore, we corroborate the motional amplitudes in the RDC-derived ensembles quantitatively with the observed amplitudes of RD and DR. The methods utilized herein can be used to directly study protein dynamics in a time range that was previously inaccessible. Significant motional amplitude in the supra-tc window has been observed using RDC measurements, and was connected to the conformational sampling for a protein in the ground