Recent Advances

Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures to represent only an averaged snapshot of the dynamic picture. While NMR relaxation measurements can help to determine local dynamics, it is difficult to detect translational or concerted motion, and only recently have significant advances been made to make it possible to acquire a more holistic representation of the dynamics and structural landscapes of proteins. Here, we briefly revisit our most recent progress in the theory and use of exact nuclear Overhauser enhancements (eNOEs) for the calculation of structural ensembles that describe their conformational space. New developments are primarily targeted at increasing the number and improving the quality of extracted eNOE distance restraints, such that the multi-state structure calculation can be applied to proteins of higher molecular weights. We then review the implications of the exact NOE to the protein dynamics and function of cyclophilin A and the WW domain of Pin1, and finally discuss our current research and future directions

Редокс-регуляция динамического характера фенотипа эндотелиоцитов кровеносных сосудов

ЭНДОТЕЛИАЛЬНЫЕ КЛЕТКИКЛЕТКИЭНДОТЕЛИЙКРОВЕНОСНЫЕ СОСУДЫФЕНОТИПГЕНЕТИЧЕСКИЕ ФЕНОМЕНЫОКИСЛИТЕЛЬНО-ВОССТАНОВИТЕЛЬНЫЕ РЕАКЦИИЭНЕРГЕТИЧЕСКИЙ ОБМЕНПЕРЕКИСНОЕ ОКИСЛЕНИЕ ЛИПИДО

Solution nuclear magnetic resonance spectroscopy of bacterial outer membrane proteins in natively excreted vesicles using engineered Escherichia coli

Gaining structural information on membrane proteins in their native lipid environment is a long-standing challenge in molecular biology. Instead, it is common to employ membrane mimetics, which has been shown to affect protein structure, dynamics, and function severely. Here, we describe the incorporation of a bacterial outer membrane protein (OmpW) into natively excreted membrane vesicles for solution nuclear magnetic resonance (NMR) spectroscopy using a mutant Escherichia coli strain with a high outer membrane vesicle (OMV) production rate. We collected NMR spectra from both vesicles containing overexpressed OmpW and vesicles from a control strain to account for the presence of physiologically relevant outer membrane proteins in vesicles and observed distinct resonance signals from OmpW. Due to the increased production of OMVs and the use of non-uniform sampling techniques we were able to obtain high-resolution 2D (HSQC) and 3D (HNCO) NMR spectra of our target protein inside its native lipid environment. While this workflow is not yet sufficient to achieve in situ structure determination, our results pave the way for further research on vesicle-based solution NMR spectroscopy

Enzyme Selectivity Fine-Tuned through Dynamic Control of a Loop

Allostery has been revealed as an essential property of all proteins. For enzymes, shifting of the structural equilibrium distribution at one site can have substantial impacts on protein dynamics and selectivity. Promising sites of remotely shifting such a distribution by changing the dynamics would be at flexible loops because relatively large changes may be achieved with minimal modification of the protein. A ligand-selective change of binding affinity to the active site of cyclophilin is presented involving tuning of the dynamics of a highly flexible loop. Binding affinity is increased upon substitution of double Gly to Ala at the hinge regions of the loop. Quenching of the motional amplitudes of the loop slightly rearranges the active site. In particular, key residues for binding Phe60 and His126 adopt a more fixed orientation in the bound protein. Our system may serve as a model system for studying the effects of various time scales of loop motion on protein function tuned by mutations

The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity : Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ1

The use of nuclear magnetic resonance chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purpose, among others, of determining affinity constants, locating binding surfaces, or differentiating between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes is often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional effort. Here, we demonstrate the importance of the sign of the chemical shift difference induced by ligand-protein interaction. We analyze the sign of the 15N/1H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ-ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift perturbation analysis is insensitive. In addition, we find a correlation between affinity and millisecond motions. Application of the methodology to Cyclophilin a, a cis-trans isomerase, reveals molecular details of peptide recognition. We consider our directionality vector chemical shift analysis as a method of choice when distinguishing the molecular origin of binding specificities of a class of similar ligands, which is often done in drug discovery

Extending the eNOE data set of large proteins by evaluation of NOEs with unresolved diagonals

The representation of a protein’s spatial sampling at atomic resolution is fundamental for understanding its function. NMR has been established as the best-suited technique toward this goal for small proteins. However, the accessible information content rapidly deteriorates with increasing protein size. We have recently demonstrated that for small proteins distance restraints with an accuracy smaller than 0.1 Å can be obtained by replacing traditional semi-quantitative Nuclear Overhauser Effects (NOEs) with exact NOEs (eNOE). The high quality of the data allowed us to calculate structural ensembles of the small model protein GB3 consisting of multiple rather than a single state. The analysis has been limited to small proteins because NOEs of spins with unresolved diagonal peaks cannot be used. Here we propose a simple approach to translate such NOEs into correct upper distance restraints, which opens access to larger biomolecules. We demonstrate that for 16 kDa cyclophilin A the collection of such restraints extends the original 1254 eNOEs to 3471.ISSN:0925-2738ISSN:1573-500

Influence of Detergent and Lipid Composition on Reconstituted Membrane Proteins for Structural Studies

Membrane proteins are frequently reconstituted in different detergents as a prerequisite to create a phospholipid environment reminiscent of their native environment. Different detergent characteristics such as their chain length and bond types could affect the structure and function of proteins. Yet, they are seldom taken into account when choosing a detergent for structural studies. Here, we explore the effect of different detergents and lipids with varying degrees of double- or single-bond composition on H-1-N-15 transverse relaxation optimized spectroscopy spectra of the outer membrane protein W (OmpW). We observed changes in nuclear magnetic resonance chemical shifts for OmpW reconstituted in micelles, bicelles, and nanodiscs, depending on their detergent/lipid composition. These results suggest that a careful evaluation of detergents is necessary, so as not to jeopardize the structure and function of the protein

Ligand binding by PDZ domains

The postsynaptic density protein-95/disks large/zonula occludens-1 (PDZ) protein domain family is one of the most common proteinprotein interaction modules in mammalian cells, with paralogs present in several hundred human proteins. PDZ domains are found in most cell types, but neuronal proteins, for example, are particularly rich in these domains. The general function of PDZ domains is to bring proteins together within the appropriate cellular compartment, thereby facilitating scaffolding, signaling, and trafficking events. The many functions of PDZ domains under normal physiological as well as pathological conditions have been reviewed recently. In this review, we focus on the molecular details of how PDZ domains bind their protein ligands and their potential as drug targets in this context

NMR resonance assignment and dynamics of profilin from Heimdallarchaeota

The origin of the eukaryotic cell is an unsettled scientific question. The Asgard superphylum has emerged as a compelling target for studying eukaryogenesis due to the previously unseen diversity of eukaryotic signature proteins. However, our knowledge about these proteins is still relegated to metagenomic data and very little is known about their structural properties. Additionally, it is still unclear if these proteins are functionally homologous to their eukaryotic counterparts. Here, we expressed, purified and structurally characterized profilin from Heimdallarchaeota in the Asgard superphylum. The structural analysis shows that while this profilin possesses similar secondary structural elements as eukaryotic profilin, it contains additional secondary structural elements that could be critical for its function and an indication of divergent evolution