Molecular Dynamics Simulation Approaches to K Channels

Membrane proteins are coded for by approximately 30% of the human genome.However, the study of them is diffi cult due to their hydrophobic nature.What are the latest techniques to tackle this? ou may be surprised to learn that membrane proteins are coded for by approximately 30% of the human genome [1].That's nearly a third of all genetic information in our cells dedicated to the production of membrane proteins.Although elusive, these proteins are critical for cellular function, especially in cell communication and transport pathways.The cause of their elusiveness can be attributed to their hydrophobic nature, leading to diffi culty in structural studies because they can't be dissolved in water and are prevented from crystallizing -a necessary step in techniques such as x-ray c rystallography.Once extracted from cell membranes, the proteins are made water-soluble only when suspended in detergents that mimic the hydrophobicity of a cell membrane.However, these are expensive and there is no 'one size fi ts all'.Detergents can also disrupt the structure and function of membrane proteins, as they inte r fe re with inter-and intramolecular proteinprotein interactions.Of ~8000 known membrane proteins found in human cells, only ~50 have a determined structure [2].With membrane proteins being implicated in many different diseases [3], including heart disease, Alzheimer's and cystic fi brosis, it is of crucial importance that structures are characterized in order for novel ideas for therapies and treatments to come to light.So, just what new approaches and techniques are being developed to tackle this issue?

In strained monoatomic chains with Lennard-Jones interactions, we revealed a stable static non-homogeneous structure appearing as a result of a certain phase transition. Positions of individual particles in this structure form an exact arithmetic progression whose difference depends on the value of the strain. For N-particle chain, this structure is characterized by one long and N-1 short interatomic distances (bonds). In the vicinity of the static structure, we found discrete breathers of new type which essentially differ from the traditional breathers in the form of Sievers-Takeno and Page modes. It is well known that these modes possess some staggered structures and demonstrate exponential decay of the particle amplitudes from the core to their tails. In contrast to such properties, our breathers are characterised by smooth decay and amplitudes of the particles form approximately a decreasing arithmetic progression. Core of these breathers is located on two particles with long bond in static structure. Our breathers demonstrate soft type of nonlinearity (the frequency decreases with increasing of amplitudes) and they are stable dynamical objects for amplitudes up to 20%-30% of interparticle distance of the strained equidistant chain. For infinitely small amplitudes these breathers tend to the above described static non-homogeneous structure. We studied dependence of their properties on amplitude, strain and the number of particles in the chain. There exist a reason to suppose that the above static and dynamical structures can exist in real monoatomic chains consisting of carbon, boron, and other atoms.

The influence of cholesterol on membrane protein structure, function, and dynamics studied by molecular dynamics simulations

Membrane proteins, such as receptors, transporters and ion channels, control the vast majority of cellular signalling and metabolite exchange processes and thus are becoming key pharmacological targets. Obtaining structural information by usage of traditional structural biology techniques is limited by the requirements for the protein samples to be highly pure and stable when handled in high concentrations and in non-native buffer systems, which is often difficult to achieve for membrane targets. Hence, there is a growing requirement for the use of hybrid, integrative approaches to study the dynamic and functional aspects of membrane proteins in physiologically relevant conditions. In recent years, significant progress has been made in the field of oxidative labelling techniques and in particular the X-ray radiolytic footprinting in combination with mass spectrometry (MS) (XF-MS), which provide residue-specific information on the solvent accessibility of proteins. In combination with both low- and high-resolution data from other structural biology approaches, it is capable of providing valuable insights into dynamics of membrane proteins, which have been difficult to obtain by other structural techniques, proving a highly complementary technique to address structure and function of membrane targets. XF-MS has demonstrated a unique capability for identification of structural waters and conformational changes in proteins at both a high degree of spatial and a high degree of temporal resolution. Here, we provide a perspective on the place of XF-MS among other structural biology methods and showcase some of the latest developments in its usage for studying water-mediated transmembrane (TM) signalling, ion transport and ligand-induced allosteric conformational changes in membrane proteins.

The Xenopus oocyte is a very powerful tool for studies of the structure and function of membrane proteins, e.g., messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional receptors and ion channels, and membrane vesicles from Torpedo electroplaques injected into oocytes fuse with the oocyte membrane and cause the appearance of functional Torpedo acetylcholine receptors and Cl(-) channels. This approach was developed further to transplant already assembled neurotransmitter receptors from human brain cells to the plasma membrane of Xenopus oocytes. Membranes isolated from the temporal neocortex of a patient, operated for intractable epilepsy, were injected into oocytes and, within a few hours, the oocyte membrane acquired functional neurotransmitter receptors to gamma-aminobutyric acid, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and glycine. These receptors were also expressed in the plasma membrane of oocytes injected with mRNA extracted from the temporal neocortex of the same patient. All of this makes the Xenopus oocyte a more useful model than it already is for studies of the structure and function of many human membrane proteins and opens the way to novel pathophysiological investigations of some human brain disorders.

STATIC STRUCTURE, DYNAMIC FORM: AN ANALYSIS OF ELLIOTT CARTER’S CONCERTO FOR ORCHESTRA KLAAS COULEMBIER INTRODUCTION LLIOTT CARTER’S CONCERTO FOR ORCHESTRA is one of his most intriguing, complex, and fascinating compositions. It is a tour de force in the organization and arrangement of different musical materials, in the domains of both pitch and rhythm. Several authors have expressed their admiration for the composition, while acknowledging that it is very difficult to penetrate. In 1989, David Harvey opened his chapter on Carter’s Concerto for Orchestra with the following statement: The Concerto for Orchestra is Carter’s richest and most complex work to date in every respect. A complete account of its material, techniques, and their realisation in the textures of the work is beyond the scope of the present study; indeed, it may be doubted that such a project is at all feasible, given the size of the work, the density of the orchestral writing, the richness of the harmonic elaborations generated by Carter’s intervallic techniques of composition , and the limitations of analysis at the present time.1 E 98 Perspectives of New Music That this work is attractive to analysts is beyond question; the trepidation with which they have approached is, however, not only the result of its multi-layered musical surface, but also, no doubt, stems from the startling number of sketches Carter generated in producing it, conveying the impression that the construction of the composition may be even more puzzling than its form. Nevertheless, to gain a deeper insight into the workings of this music, the sketches appear to be as necessary as they are daunting. Jonathan Bernard concluded his 1983 article in Music Analysis with the remark that it would be impossible to make a fully comprehensive analysis of this composition. Dealing with the huge expanses of Carter’s scores . . . is still not easy. To retrace the steps of a composer who produces thousands of pages of sketches and works for thousands of hours in the course of writing a piece is likely to be a formidable undertaking, to say the least. . . . The prospect of reading, much less writing, a so-called “complete analysis” carried out according to the methods presented here is truly fearsome to contemplate. Eventually, perhaps, someone will have a bright idea that will make everything seem much simpler. Until then we can only have faith in the music, continue to analyse, and hope for the best.2 Rather than try to be that person with the bright idea, I want to contribute to the understanding of this composition by focusing on its overall temporal and dramatic organization. Therefore, in dealing with the more than 3000 pages of sketches that I studied during a weeklong stay at the Paul Sacher Foundation, I have kept my focus here exclusively on rhythmic sketches and temporal calculations. In that respect the following analysis complements existing literature in which pitch organization has often been the focal point. Despite the dependence on sketches, this analysis is not aimed at a mere reconstruction of the compositional process.3 By revealing the intricate relation between the rigid and static background structures and their more supple and subtle surface manifestations, this analysis tries to show which strategies and methods Carter applied to achieve such a compelling dramatic and dynamic musical discourse. THE CONCERTO FOR ORCHESTRA IN LITERATURE: AN OVERVIEW Apart from a chapter in David Harvey’s dissertation,4 there are no exhaustive, start-to-finish analyses of the Concerto for Orchestra. Static Structure, Dynamic Form 99 David Schiff gives a comprehensible overview of the composition in The Music of Elliott Carter.5 Jonathan Bernard has returned to the composition on several occasions, dealing with pitch structures in his article on “spatial sets,”6 or with the relationship between the literary source of the composition in “Poem as Non-Verbal Text.”7 Several publications of the Paul Sacher Foundation also include descriptions of the composition’s elaborate sketch resources available in their collection.8 Scholarly literature on Carter’s music in general has regained new energy in the last decade, particularly since the composer’s centennial celebration in 2008. Recent publications often consolidate important studies (such as Jonathan Bernard...

Computational Design of Membrane Proteins

GLIC is a homopentameric proton-gated, prokaryotic homologue of the Cys loop receptor family of neurotransmitter-gated ion channels. Recently, crystal structures of GLIC hypothesized to represent an open channel state were published. To explore the channel structure in functional GLIC channels, we tested the ability of p-chloromercuribenzenesulfonate to react with 30 individual cysteine substitution mutants in and flanking the M2 channel-lining segment in the closed state (pH 7.5) and in a submaximally activated state (pH 5.0). Nine mutants did not tolerate cysteine substitution and were not functional. From positions 10' to 27', p-chloromercuribenzenesulfonate significantly modified the currents at pH 7.5 and 5.0 in all mutants except H234C (11'), I235C (12'), V241C (18'), T243C (20'), L245C (22'), and Y250C (27'), which were not functional, except for 12'. Currents for P246C (23') and K247C (24') were only significantly altered at pH 5.0. The reaction rates were all >1000 m(-1) s(-1). The reactive residues were more accessible in the activated than in the resting state. We infer that M2 is tightly associated with the adjacent transmembrane helices at the intracellular end but is more loosely packed from 10' to the extracellular end than the x-ray structures suggest. We infer that the charge selectivity filter is in the cytoplasmic half of the channel. We also show that below pH 5.0, GLIC desensitizes on a time scale of minutes and infer that the crystal structures may represent a desensitized state.

The emergence of deep learning, particularly AlphaFold, has revolutionized static protein structure prediction, marking a transformative milestone in structural biology. However, protein function is not solely determined by static three-dimensional structures but is fundamentally governed by dynamic transitions between multiple conformational states. This shift from static to multi-state representations is crucial for understanding the mechanistic basis of protein function and regulation. This review outlines the fundamental concepts of protein dynamic conformations, surveys recent computational advances in modeling these dynamics in the post-AlphaFold era, and highlights key challenges, including data limitations, methodological constraints, and evaluation metrics. We also discuss potential strategies to address these challenges and explore future research directions to deepen our understanding of protein dynamics and their functional implications. This work aims to provide insights and perspectives to facilitate the ongoing development of protein conformation studies in the era of artificial intelligence-driven structural biology.

From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

Transient receptor potential (TRP) cation channels are unique cellular sensors that are involved in multiple cellular functions, ranging from transduction of sensory signals to the regulation of Ca2+ and Mg2+ homoeostasis. Malfunctioning of TRP channels is now recognized as the cause of several hereditary and acquired human diseases. At the time of cloning of the first Drosophila TRP channel, a close connection between gating and phosphatidylinositol phosphates (PIPs) was already recognized. In this review, we summarize current knowledge about the mechanisms of interaction between TRP channels and PIPs, and discuss the possible functional implications of TRP–PIP interactions to human physiology and pathophysiology.

Symmetry, Selectivity, and the 2003 Nobel Prize

Computational analyses on the structure-function relation inion channels

TASK channels are background K+ channels that contribute to the resting conductance in many neurons. A key feature of TASK channels is the reversible inhibition by Gq-coupled receptors, thereby mediating the dynamic regulation of neuronal activity by modulatory transmitters. The mechanism that mediates channel inhibition is not fully understood. While it is clear that activation of Gαq is required, the immediate signal for channel closure remains controversial. Experimental evidence pointed to either phospholipase C (PLC)-mediated depletion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) as the cause for channel closure or to a direct inhibitory interaction of active Gαq with the channel. Here, we address the role of PI(4,5)P2 for G-protein-coupled receptor (GPCR)-mediated TASK inhibition by using recently developed genetically encoded tools to alter phosphoinositide (PI) concentrations in the living cell.When expressed in CHO cells, TASK-1- and TASK-3-mediated currents were not affected by depletion of plasma membrane PI(4,5)P2 either via the voltage-activated phosphatase Ci-VSP or via chemically triggered recruitment of a PI(4,5)P2-5'-phosphatase. Depletion of both PI(4,5)P2 and PI(4)P via membrane recruitment of a novel engineered dual-specificity phosphatase also did not inhibit TASK currents. In contrast, each of these methods produced robust inhibition of the bona fide PI(4,5)P2-dependent channel KCNQ4. Efficient depletion of PI(4,5)P2 and PI(4)P was further confirmed with a fluorescent phosphoinositide sensor. Moreover, TASK channels recovered normally from inhibition by co-expressed muscarinic M1 receptors when resynthesis of PI(4,5)P2 was prevented by depletion of cellular ATP. These results demonstrate that TASK channel activity is independent of phosphoinositide concentrations within the physiological range. Consequently, Gq-mediated inhibition of TASK channels is not mediated by depletion of PI(4,5)P2.

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels