Normal Mode Analysis: A Tool for Better Understanding Protein Flexibility and Dynamics with Application to Homology Models

Molecular dynamics (MD) and normal mode analysis (NMA) are very useful methods for characterizing various dynamic aspects of biological macromolecules. In comparison to MD, NMA is computationally less expensive which facilitates the quick and systematic investigation of protein flexibility and dynamics even for large proteins and protein complexes, whose structure was obtained experimentally or in silico. In particular, NMA can be used to describe the flexible states adopted by a protein around an equilibrium position. These states have been repeatedly shown to have biological relevance and functional significance. This chapter briefly characterizes NMA and describes the elastic network model, a schematic model of protein shape used to decrease the computational cost of this method. Finally, we will describe the applications of this technique to several large proteins and their complexes as well as its use in enhancing protein homology modeling

Structure and Function of the Human Ryanodine Receptors and Their Association with Myopathies—Present State, Challenges, and Perspectives

Cardiac arrhythmias are serious, life-threatening diseases associated with the dysregulation of Ca2+ influx into the cytoplasm of cardiomyocytes. This dysregulation often arises from dysfunction of ryanodine receptor 2 (RyR2), the principal Ca2+ release channel. Dysfunction of RyR1, the skeletal muscle isoform, also results in less severe, but also potentially life-threatening syndromes. The RYR2 and RYR1 genes have been found to harbor three main mutation “hot spots”, where mutations change the channel structure, its interdomain interface properties, its interactions with its binding partners, or its dynamics. In all cases, the result is a defective release of Ca2+ ions from the sarcoplasmic reticulum into the myocyte cytoplasm. Here, we provide an overview of the most frequent diseases resulting from mutations to RyR1 and RyR2, briefly review some of the recent experimental structural work on these two molecules, detail some of the computational work describing their dynamics, and summarize the known changes to the structure and function of these receptors with particular emphasis on their N-terminal, central, and channel domains

Normal Mode Analysis as a Routine Part of a Structural Investigation

Normal mode analysis (NMA) is a technique that can be used to describe the flexible states accessible to a protein about an equilibrium position. These states have been shown repeatedly to have functional significance. NMA is probably the least computationally expensive method for studying the dynamics of macromolecules, and advances in computer technology and algorithms for calculating normal modes over the last 20 years have made it nearly trivial for all but the largest systems. Despite this, it is still uncommon for NMA to be used as a component of the analysis of a structural study. In this review, we will describe NMA, outline its advantages and limitations, explain what can and cannot be learned from it, and address some criticisms and concerns that have been voiced about it. We will then review the most commonly used techniques for reducing the computational cost of this method and identify the web services making use of these methods. We will illustrate several of their possible uses with recent examples from the literature. We conclude by recommending that NMA become one of the standard tools employed in any structural study

Glucose Oxidase, an Enzyme “Ferrari”: Its Structure, Function, Production and Properties in the Light of Various Industrial and Biotechnological Applications

Glucose oxidase (GOx) is an important oxidoreductase enzyme with many important roles in biological processes. It is considered an “ideal enzyme” and is often called an oxidase “Ferrari” because of its fast mechanism of action, high stability and specificity. Glucose oxidase catalyzes the oxidation of β-d-glucose to d-glucono-δ-lactone and hydrogen peroxide in the presence of molecular oxygen. d-glucono-δ-lactone is sequentially hydrolyzed by lactonase to d-gluconic acid, and the resulting hydrogen peroxide is hydrolyzed by catalase to oxygen and water. GOx is presently known to be produced only by fungi and insects. The current main industrial producers of glucose oxidase are Aspergillus and Penicillium. An important property of GOx is its antimicrobial effect against various pathogens and its use in many industrial and medical areas. The aim of this review is to summarize the structure, function, production strains and biophysical and biochemical properties of GOx in light of its various industrial, biotechnological and medical applications

Disease-associated mutations alter the dynamic motion of the N-terminal domain of the human cardiac ryanodine receptor

The human cardiac ryanodine receptor (hRyR2), the ion channel responsible for the release of Ca$^{2+}$ ions from the sarcoplasmic reticulum into the cytosol, plays an important role in cardiac muscle contraction. Mutations to this channel are associated with inherited cardiac arrhythmias. These mutations appear to cluster in distinct parts of the N-terminal, central and C-terminal areas of the channel. Here, we used molecular dynamics simulation to examine the effects three disease-associated mutations to the N-terminal region, R414L, I419F and R420W, have on the dynamics of a model of residues 1–655 of hRyR2. We find that the R414L and I419F mutations diminish the overall amplitude of motion without greatly changing the direction of motion of the individual domains, whereas R420W both enhances the amplitude and changes the direction of motion. Based on these results, we hypothesize that R414L and I419F hinder channel closing, whereas R420W may enhance channel opening. Overall, it appears that the wild-type protein possesses a moderate level of flexibility which allows the gate to close and not easily open without an opening signal. These mutations, however, disrupt this balance by making the gate either too rigid or too loose, causing closing to become difficult or less effective. Small-angle X-ray scattering studies of the same 1–655 residue fragment are in agreement with the molecular dynamics results and also suggest that the rest of the protein is needed to keep the entire domain properly folded

Eu3+ detects two functionally distinct luminal Ca2+ binding sites in ryanodine receptors

Ryanodine receptors (RyRs) are Ca2+ release channels, gated by Ca2+ in the cytosol and the sarcoplasmic reticulum lumen. Their regulation is impaired in certain cardiac and muscle diseases. Although a lot of data is available on the luminal Ca2+ regulation of RyR, its interpretation is complicated by the possibility that the divalent ions used to probe the luminal binding sites may contaminate the cytoplasmic sites by crossing the channel pore. In this study, we used Eu3+, an impermeable agonist of Ca2+ binding sites, as a probe to avoid this complication and to gain more specific information about the function of the luminal Ca2+ sensor. Single-channel currents were measured from skeletal muscle and cardiac RyRs (RyR1 and RyR2) using the lipid bilayer technique. We show that RyR2 is activated by the luminal addition of Ca2+, whereas RyR1 is inhibited. These results were qualitatively reproducible using Eu3+. The luminal regulation of RyR1 carrying a mutation associated with malignant hyperthermia was not different from that of the wild-type. RyR1 inhibition by Eu3+ was extremely voltage dependent, whereas RyR2 activation did not depend on the membrane potential. These results suggest that the RyR1 inhibition site is in the membrane’s electric field (channel pore), whereas the RyR2 activation site is outside. Using in silico analysis and previous results, we predicted putative Ca2+ binding site sequences. We propose that RyR2 bears an activation site, which is missing in RyR1, but both isoforms share the same inhibitory Ca2+ binding site near the channel gate