Insights into internal dynamics of 6-phosphogluconolactonase from Trypanosoma brucei studied by nuclear magnetic resonance and molecular dynamics

Nuclear magnetic resonance is used to investigate the backbone dynamics in 6-phosphogluconolactonase from Trypanosoma brucei (Tb6PGL) with (holo-) and without (apo-) 6-phosphogluconic acid as ligand. Relaxation data were analyzed using the model-free approach and reduced spectral density mapping. Comparison of predictions, based on 77 ns molecular dynamics simulations, with the observed relaxation rates gives insight into dynamical properties of the protein and their alteration on ligand binding. Data indicate dynamics changes in the vicinity of the binding site. More interesting is the presence of perturbations located in remote regions of this well-structured globular protein in which no large-amplitude motions are involved. This suggests that delocalized changes in dynamics that occur upon binding could be a general feature of proteintarget interactions. Proteins 2012; (c) 2011 Wiley Periodicals, Inc.

Study of fast exchanging processes by NMR Spectroscopy

Estel Canet Martinez

Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful and informative methods to probe molecular dynamics. Specifically, chemical exchange is an important phenomenon and one of the earliest and most vigorously investigated in NMR spectroscopy. Its effects can be observed in many NMR spectra; lines in a spectrum are broadened or averaged because the nuclei are exchanging magnetic environments. A wide variety of experimental methods exists to determine exchange rates of protons that hop between amino acids and water in the slow or intermediate exchange regime. However, so far there is a lack of methods to investigate hydrogen exchange in the fast exchange regime. In this thesis, we focus on the determination of very fast chemical exchange rates from the effects of scalar relaxation on a coupled nucleus. We quantify the effects of scalar relaxation caused by exchanging hydrogens, by detecting the decay of a 15N coherence under a multiple-refocusing Carr-Purcell-Meiboom-Gill (CPMG) pulse train in the presence or absence of hydrogen decoupling. In particular, we have adapted to the case of deuterium a method that was originally designed to study the exchange rate of the indole proton of tryptophan in water as a function of pH and temperature. We will present the exchange rates of the indole deuterium in tryptophan with solvent heavy water. After that, the proton exchanges rates have been compared with the deuterium exchange rates in order to describe the kinetic isotope effect. In a similar manner, we have quantified the proton exchange rates in pure water by using multiple refocusing of transverse 17O magnetization as a function of pH and temperature. Such measurements are widely used to obtain valuable information about molecular dynamics and structure of proteins, protein complexes and nucleic acids. The knowledge of the kinetic isotope effect may contribute to the characterization of reaction mechanisms and other dynamic parameters that can give insight into the stability of hydrogen bonded secondary structures in biomolecules. This information could form a foundation for a more thorough theoretical or modeling development of a number of long and outstanding fundamental questions. In the second part of the thesis, we combine fast exchange with dissolution dynamic nuclear polarization (D-DNP) to study intrinsically disordered proteins (IDPs). IDPs constitute a very large and functionally important class of proteins that lack stable secondary and tertiary structures. As a consequence of the broad dynamics and flexibility of IDPs, and specially under physiological conditions, the NMR spectra of IDPs typically show a strong signal overlap and broadening due to proton exchange. We present a method to overcome these limitations by means of hyperpolarized HDO produced by D-DNP. We could study the effects of conformational adaptations in Osteopontin (OPN), a metastasis- associated intrinsically disordered protein (IDP) by chemical exchange. The magnetization from hyperpolarized water is transferred to the rapidly exchanging protons of the solvent-exposed residues of the protein. The exchange with hyperpolarized water boosts the signals of fast exchanging residues above the detection limits. This allows one to follow individual resonances and draw conclusions regarding conformational adaptations due to ligand binding under physiological conditions. Thus, with our approach, exchange with the solvent is turned into an advantage

EPFL2017

Picometer Resolution Structure of the Coordination Sphere in the Metal-Binding Site in a Metalloprotein by NMR

Andrea Bertarello, David Lyndon Emsley

Most of our understanding of chemistry derives from atomic-level structures obtained with single-crystal X-ray diffraction. Metal centers in X-ray structures of small organometallic or coordination complexes are often extremely well-defined, with errors in the positions on the order of 10(-4)-10(-5) A. Determining the metal coordination geometry to high accuracy is essential for understanding metal center reactivity, as even small structural changes can dramatically alter the metal activity. In contrast, the resolution of X-ray structures in proteins is limited typically to the order of 10(-1) angstrom. This resolution is often not sufficient to develop precise structure-activity relations for the metal sites in proteins, because the uncertainty in positions can cover all of the known ranges of bond lengths and bond angles for a given type of metal complex. Here we introduce a new approach that enables the determination of a high-definition structure of the active site of a metalloprotein from a powder sample, by combining magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, tailored radio frequency (RF) irradiation schemes, and computational approaches. This allows us to overcome the "blind sphere" in paramagnetic proteins, and to observe and assign H-1, C-13, and N-15 resonances for the ligands directly coordinating the metal center. We illustrate the method by determining the bond lengths in the structure of the Co-II coordination sphere at the core of human superoxide dismutase 1 (SOD) with 0.7 pm precision. The coordination geometry of the resulting structure explains the nonreactive nature of the Co-II/Zn-II centers in these proteins, which allows them to play a purely structural role.

2020

Chemical exchange in proteins

Mariachiara Verde

Nuclear Magnetic Resonance (NMR) spectroscopy offers many ways to investigate dynamic properties of molecules. A wide variety of experimental techniques can probe molecular dynamics on time scales that range from 10-12 to 103 seconds. Many lines of evidence point to the existence of large scale dynamics on milli- to microsecond time scale that are essential to molecular function. Those conformational motions can give rise to chemical exchange effects. So far in the literature two methods have been mainly used to study such processes: CPMG echo trains and R1ρ spin-lock relaxation experiments. The former is suited to investigate processes on the millisecond time scale, whereas R1ρ can probe processes ranging from milliseconds down to ca. microseconds. CPMG and R1ρ studies of the relaxation rates of single quantum (SQ) coherences can be complemented by multiple quantum coherence (MQ) coherences experiments which can provide information whether two (or more) spins are affected simultaneously by chemical exchange. The goal of this thesis is to characterize chemical exchange in proteins by new (Heteronuclear Double Resonance, HDR) and existing (relaxation compensated CPMG) methods. The presence of chemical exchange has been identified in APO-rMUP using the classical CPMG experiment on single quantum 15N magnetization, whereas in HOLO-rMUP no presence of chemical exchange has been found. Most of the thesis is dedicated to the analysis and the application of a new method, HDR, which is designed to determine the cross-relaxation rates between MQ operators. In fact, in heteronuclear systems, correlated chemical exchange contributes to cross-relaxation between MQ coherences 2IxSx and 2IySy. This method, based on MLEV-32 and WALTZ-16 used in double resonance mode, is designed to effectively preserve all relevant MQ coherences simultaneously so that the interconversion between MQ operators can only arise through cross-relaxation. This is an essential requirement if we want to measure, for example, small cross-relaxation rates between MQ operators. We have treated the effects of coherent evolution during the HDR sequences by numerical simulations and Average Hamiltonian Theory (AHT) showing that, under most conditions, the dynamics of MQ coherences is not affected by coherent evolution. This result is proved experimentally on the 15N-1H pair of selectively deuterated tBoc-glycine. The effect of relaxation during HDR sequences leads to an effective relaxation superoperator which is explored by numerical simulations and predicted by Average Liouvillian Theory (ALT). Finally the HDR MLEV-32 sequence is applied to amide 1H-15N pairs in ubiquitin and KIX proteins. In the case of ubiquitin the HDR MLEV-32 experiments and the numerical simulations run with parameters obtained from the literature are in good agreement, suggesting that HDR sequences could be used as a tool to obtain information about the dynamics of the system.