Robust structure-based resonance assignment for functional protein studies by NMR

High-throughput functional protein NMR studies, like protein interactions or dynamics, require an automated approach for the assignment of the protein backbone. With the availability of a growing number of protein 3D structures, a new class of automated approaches, called structure-based assignment, has been developed quite recently. Structure-based approaches use primarily NMR input data that are not based on J-coupling and for which connections between residues are not limited by through bonds magnetization transfer efficiency. We present here a robust structure-based assignment approach using mainly HN–HN NOEs networks, as well as 1H–15N residual dipolar couplings and chemical shifts. The NOEnet complete search algorithm is robust against assignment errors, even for sparse input data. Instead of a unique and partly erroneous assignment solution, an optimal assignment ensemble with an accuracy equal or near to 100% is given by NOEnet. We show that even low precision assignment ensembles give enough information for functional studies, like modeling of protein-complexes. Finally, the combination of NOEnet with a low number of ambiguous J-coupling sequential connectivities yields a high precision assignment ensemble. NOEnet will be available under: http://www.icsn.cnrs-gif.fr/download/nmr

Mapping the encounter state of a transient protein complex by PRE NMR spectroscopy

Many biomolecular interactions proceed via a short-lived encounter state, consisting of multiple, lowly-populated species invisible to most experimental techniques. Recent development of paramagnetic relaxation enhancement (PRE) nuclear magnetic resonance (NMR) spectroscopy has allowed to directly visualize such transient intermediates in a number of protein-protein and protein-DNA complexes. Here we present an analysis of the recently published PRE NMR data for a protein complex of yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP). First, we describe a simple, general method to map out the spatial and temporal distributions of binding geometries constituting the Cc-CcP encounter state. We show that the spatiotemporal mapping provides a reliable estimate of the experimental coverage and, at higher coverage levels, allows to delineate the conformational space sampled by the minor species. To further refine the encounter state, we performed PRE-based ensemble simulations. The generated solutions reproduce well the experimental data and lie within the allowed regions of the encounter maps, confirming the validity of the mapping approach. The refined encounter ensembles are distributed predominantly in a region encompassing the dominant form of the complex, providing experimental proof for the results of classical theoretical simulations

A NMR investigation on the interactions of the alpha-oligomeric form of the M13 coat protein with lipids, which mimic the Escherichia coli inner membrane.

The interaction of the M13 bacteriophage major coat protein in the alpha-oligomeric form with specifically deuterated phospholipid headgroups which mimic the Escherichia coli inner membrane, has been studied using NMR methods. As can be seen from the deuterium NMR spectra obtained with headgroup trimethyl deuterated DOPC, the coat protein in the alpha-oligomeric form does not give rise to trapped lipids as observed with M13 coat protein in the beta-polymeric form (Van Gorkom et al. (1990) Biochemistry 29, 3828-3834). The quadrupolar splittings of the alpha headgroup methylene deuterons of deuterated phosphatidylcholine and phosphatidylethanolamine decrease, whereas the quadrupolar splittings of the beta headgroup methylene deuterons of the two lipids increase with increasing protein content. All deuterated segments in the phosphatidylglycerol headgroup show the same relative decrease of the NMR quadrupolar splittings. These results are interpreted in terms of a change in torsion angles of the methylene groups, induced by positive charges, probably lysine residues of the protein at the membrane surface. For all lipid bilayer compositions studied the head-group perturbations are similar. It is concluded that there is no strong specific interaction between one of the lipid types examined and the M13 coat protein. From the spin-spin (T2e) relaxation time and spin-lattice (T1z) relaxation time of all deuterated lipids it is concluded that at the bilayer surface only slow motions are affected by the M13 coat protein

Following protein folding in real time using NMR spectroscopy.

The refolding of apo bovine alpha-lactalbumin has been monitored in real time by NMR spectroscopy following rapid in situ dilution of a chemically denatured state. By examining individual resonances in the time-resolved NMR spectra, the native state has been shown to emerge in a cooperative manner from an intermediate formed in the dead-time of the experiments. The kinetics of folding to the native state are closely similar to those observed by stopped-flow fluorescence and near-UV circular dichroism. The NMR spectrum of the transient intermediate resembles closely that of the well characterized stable molten globule state formed at low pH. The results suggest that NMR can play a key role in describing at an atomic level the structural transitions occurring during protein folding

Rapid oligomer formation of human muscle acylphosphatase induced by heparan sulfate

Many human diseases are caused by the conversion of proteins from their native state into amyloid fibrils that deposit in the extracellular space. Heparan sulfate, a component of the extracellular matrix, is universally associated with amyloid deposits and promotes fibril formation. The formation of cytotoxic prefibrillar oligomers is challenging to study because of its rapidity, transient appearance and the heterogeneity of species generated. The process is even more complex with agents such as heparan sulfate. Here we have used a stopped-flow device coupled to turbidometry detection to monitor the rapid conversion of human muscle acylphosphatase into oligomers with varying heparan sulfate and protein concentrations. We also analyzed mutants of the 15 basic amino acids of acylphosphatase, identifying the residues primarily involved in heparan sulfate-induced oligomerization of this protein and tracing the process with unprecedented molecular detail

Augmented and virtual reality in anatomical education – a systematic review

Learning anatomy traditionally has depended on traditional techniques like human cadaveric dissection and the use of textbooks. As technology advances at an ever-rapid speed, there are revolutionary ways to learn anatomy. A number of technologies, techniques and methodologies are utilised in anatomical education, but ones specifically receiving a lot of interest and traction is that of augmented reality and virtual reality. Although there has been a surge in interest in the use of these technologies, the literature is sparse in terms of its evaluation as to the effectiveness of such tools. Therefore, the purpose of this study is to examine in greater detail the literature specifically to see what the best practice in this field could be. By undertaking a systematic review using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we searched for articles in both Web of Science and PubMed. Using the terms “augmented reality and teaching anatomy” yielded 88 articles. We then used “virtual reality and teaching anatomy” which resulted in 200 articles. We examined these articles, including that on augmented reality and virtual reality used to teach anatomy to undergraduate and postgraduate students, residents, dentistry, nursing and veterinary students. Articles were excluded if they were systematic reviews, literature reviews, review articles, news articles, articles not written in English and any literature that presented how a virtual model was created without the evidence of students testing it. The inclusion and exclusion criteria for virtual reality were the same as augmented reality. In addition, we examined the articles to identify if they contained data which was quantitative, qualitative or both. The articles were further separated into those which were pro, neutral or against for the use of these digital technologies. Of the 288 articles, duplicate articles totalling 67 were removed and 134 articles were excluded according to our exclusion criteria. Of the 31 articles related to augmented reality, 30 were pro, one neutral and no articles against the use of this technology. Fifty-six articles related to virtual reality were categorised resulted in 45 pro, eight neutral and three against the use of this technology. Overall, the results indicate most articles identified related to both virtual and augmented reality were for the use of those technologies, than neutral or against. This systemic review highlights the recent advances of both augmented reality and virtual reality to implementing the technology into the anatomy course

Refolding of Ribonuclease A monitored by real-time photo-CIDNP NMR spectroscopy

Photo-CIDNP NMR spectroscopy is a powerful method for investigating the solvent accessibility of histi- dine, tyrosine and tryptophan residues in a protein. When coupled to real-time NMR, this technique allows changes in the environments of these residues to be used as a probe of protein folding. In this paper we describe experiments performed to monitor the refolding of ribonuclease A fol- lowing dilution from a high concentration of chemical denaturant. These experiments provide a good example of the utility of this technique which provides information that is difficult to obtain by other biophysical methods. Real- time photo-CIDNP measurements yield residue-specific kinetic data pertaining to the folding reaction, interpreted in terms of current knowledge of the folding of bovine pancreatic ribonuclease A