Membrane Protein Properties Revealed through Data-Rich Electrostatics Calculations.

The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem, including full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane-induced pKa shifts, calculation of non-polar energies, and command-line scripting for large-scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane, potentially revealing interesting functional information

The Sixth Transmembrane Segment Is a Major Gating Component of the TMEM16A Calcium-Activated Chloride Channel.

Calcium-activated chloride channels (CaCCs) formed by TMEM16A or TMEM16B are broadly expressed in the nervous system, smooth muscles, exocrine glands, and other tissues. With two calcium-binding sites and a pore within each monomer, the dimeric CaCC exhibits voltage-dependent calcium sensitivity. Channel activity also depends on the identity of permeant anions. To understand how CaCC regulates neuronal signaling and how CaCC is, in turn, modulated by neuronal activity, we examined the molecular basis of CaCC gating. Here, we report that voltage modulation of TMEM16A-CaCC involves voltage-dependent occupancy of calcium- and anion-binding site(s) within the membrane electric field as well as a voltage-dependent conformational change intrinsic to the channel protein. These gating modalities all critically depend on the sixth transmembrane segment

Dynamic hydraulic fluid stimulation regulated intramedullary pressure ☆

Physical signals within the bone, i.e. generated from mechanical loading, have the potential to initiate skeletal adaptation. Strong evidence has pointed to bone fluid flow (BFF) as a media between an external load and the bone cells, in which altered velocity and pressure can ultimately initiate the mechanotransduction and the remodeling process within the bone. Load-induced BFF can be altered by factors such as intramedullary pressure (ImP) and/ or bone matrix strain, mediating bone adaptation. Previous studies have shown that BFF induced by ImP alone, with minimum bone strain, can initiate bone remodeling. However, identifying induced ImP dynamics and bone strain factor in vivo using a non-invasive method still remains challenging. To apply ImP as a means for alteration of BFF, it was hypothesized that non-invasive dynamic hydraulic stimulation (DHS) can induce local ImP with minimal bone strain to potentially elicit osteogenic adaptive responses via bone-muscle coupling. The goal of this study was to evaluate the immediate effects on local and distant ImP and strain in response to a range of loading frequencies using DHS. Simultaneous femoral and tibial ImP and bone strain values were measured in three 15-month-old female Sprague Dawley rats during DHS loading on the tibia with frequencies of 1 Hz to 10 Hz. DHS showed noticeable effects on ImP induction in the stimulated tibia in a nonlinear fashion in response to DHS over the range of loading frequencies, where they peaked at 2 Hz. DHS at various loading frequencies generated minimal bone strain in the tibiae. Maximal bone strain measured at all loading frequencies was less than 8 με. No detectable induction of ImP or bone strain was observed in the femur. This study suggested that oscillatory DHS may regulate the local fluid dynamics with minimal mechanical strain in the bone, which serves critically in bone adaptation. These results clearly implied DHS's potential as an effective, non-invasive intervention for osteopenia and osteoporosis treatments. © 2013 Published by Elsevier Inc. Introduction Aging or functional disuse of the bone can subsequently create a number of physiological or pathophysiological changes in the skeleton of the affected subjects (e.g. elderly, long-term bed-rest patients, and astronauts who participate in long-duration spaceflight missions), leading to conditions such as osteopenia As a potent regulator in bone adaptation, bone fluid flow (BFF) with altered velocity or pressure acts as a communication media between an external load and the bone cells, which then regulate bone remodeling Therefore, ImP-induced BFF provides a great potential in developing novel mechanical stimuli as countermeasures for disuse bone loss. Previous in vivo study using oscillatory electrical muscle stimulation (MS) in a hindlimb suspension (HLS) functional disuse rat model has demonstrated that oscillatory MS-induced muscle contraction can generate ImP and bone strain to mitigate disuse osteopeni

TMEM16 scramblases facilitate lipid permeation by membrane bending and lipid network interactions

The TMEM16 membrane protein family consists of both scramblases and ion channels involved in ol- faction, nociception, smooth muscle contraction, and blood coagulation. A scramblase is a membrane protein that conducts lipid headgroups from one leaflet to the other in a non specific and energy indepen- dent manner. The ion conduction pathway and the lipid conduction pathway in TMEM16 proteins are not known. Some researchers believe both may permeate the same path, but the precise mechanism of ion and lipid permeation is not clearly understood. This thesis is composed of two parts. In the first part, I develop methods that utilize continuum elasticity theory, electrostatics, and solvent accessible surface area calculations to explore resident protein interaction with the lipid bilayer. I test these methods on the well studied proteins, TRPV1 and Gramicidin, while adding insight to previous work. In the sec- ond part, I combine these methods with convention molecular dynamics simulations to understand lipid permeation in the Nectria hematococca TMEM16 scramblase. I identified specific residues on the pro- tein surface that are responsible for scrambling, and homologous positions in the mammalian TMEM16 family members may be promising pharmaceutical targets

TMEM16 scramblases facilitate lipid permeation by membrane bending and lipid network interactions

The TMEM16 membrane protein family consists of both scramblases and ion channels involved in ol- faction, nociception, smooth muscle contraction, and blood coagulation. A scramblase is a membrane protein that conducts lipid headgroups from one leaflet to the other in a non specific and energy indepen- dent manner. The ion conduction pathway and the lipid conduction pathway in TMEM16 proteins are not known. Some researchers believe both may permeate the same path, but the precise mechanism of ion and lipid permeation is not clearly understood. This thesis is composed of two parts. In the first part, I develop methods that utilize continuum elasticity theory, electrostatics, and solvent accessible surface area calculations to explore resident protein interaction with the lipid bilayer. I test these methods on the well studied proteins, TRPV1 and Gramicidin, while adding insight to previous work. In the sec- ond part, I combine these methods with convention molecular dynamics simulations to understand lipid permeation in the Nectria hematococca TMEM16 scramblase. I identified specific residues on the pro- tein surface that are responsible for scrambling, and homologous positions in the mammalian TMEM16 family members may be promising pharmaceutical targets

Atomistic insight into lipid translocation by a TMEM16 scramblase

The transmembrane protein 16 (TMEM16) family of membrane proteins includes both lipid scramblases and ion channels involved in olfaction, nociception, and blood coagulation. The crystal structure of the fungal Nectria haematococca TMEM16 (nhTMEM16) scramblase suggested a putative mechanism of lipid transport, whereby polar and charged lipid headgroups move through the low-dielectric environment of the membrane by traversing a hydrophilic groove on the membrane-spanning surface of the protein. Here, we use computational methods to explore the membrane-protein interactions involved in lipid scrambling. Fast, continuum membrane-bending calculations reveal a global pattern of charged and hydrophobic surface residues that bends the membrane in a large-amplitude sinusoidal wave, resulting in bilayer thinning across the hydrophilic groove. Atomic simulations uncover two lipid headgroup-interaction sites flanking the groove. The cytoplasmic site nucleates headgroup-dipole stacking interactions that form a chain of lipid molecules that penetrate into the groove. In two instances, a cytoplasmic lipid interdigitates into this chain, crosses the bilayer, and enters the extracellular leaflet, and the reverse process happens twice as well. Continuum membrane-bending analysis carried out on homology models of mammalian homologs shows that these family members also bend the membrane-even those that lack scramblase activity. Sequence alignments show that the lipid-interaction sites are conserved in many family members but less so in those with reduced scrambling ability. Our analysis provides insight into how large-scale membrane bending and protein chemistry facilitate lipid permeation in the TMEM16 family, and we hypothesize that membrane interactions also affect ion permeation

A multiscale model of mechanotransduction by the ankyrin chains of the NOMPC channel.

Our senses of touch and hearing are dependent on the conversion of external mechanical forces into electrical impulses by the opening of mechanosensitive channels in sensory cells. This remarkable feat involves the conversion of a macroscopic mechanical displacement into a subnanoscopic conformational change within the ion channel. The mechanosensitive channel NOMPC, responsible for hearing and touch in flies, is a homotetramer composed of four pore-forming transmembrane domains and four helical chains of 29 ankyrin repeats that extend 150 Å into the cytoplasm. Previous work has shown that the ankyrin chains behave as biological springs under extension and that tethering them to microtubules could be involved in the transmission of external forces to the NOMPC gate. Here we combine normal mode analysis (NMA), full-atom molecular dynamics simulations, and continuum mechanics to characterize the material properties of the chains under extreme compression and extension. NMA reveals that the lowest-frequency modes of motion correspond to fourfold symmetric compression/extension along the channel, and the lowest-frequency symmetric mode for the isolated channel domain involves rotations of the TRP domain, a putative gating element. Finite element modeling reveals that the ankyrin chains behave as a soft spring with a linear, effective spring constantof 22 pN/nm for deflections ≤15 Å. Force-balance analysis shows that the entire channel undergoes rigid body rotation during compression, and more importantly, each chain exerts a positive twisting moment on its respective linker helices and TRP domain. This torque is a model-independent consequence of the bundle geometry and would cause a clockwise rotation of the TRP domain when viewed from the cytoplasm. Force transmission to the channel for compressions >15 Å depends on the nature of helix-helix contact. Our work reveals that compression of the ankyrin chains imparts a rotational torque on the TRP domain, which potentially results in channel opening

Continuum descriptions of membranes and their interaction with proteins: Towards chemically accurate models

Biological membranes deform in response to resident proteins leading to a coupling between membrane shape and protein localization. Additionally, the membrane influences the function of membrane proteins. Here we review contributions to this field from continuum elastic membrane models focusing on the class of models that couple the protein to the membrane. While it has been argued that continuum models cannot reproduce the distortions observed in fully-atomistic molecular dynamics simulations, we suggest that this failure can be overcome by using chemically accurate representations of the protein. We outline our recent advances along these lines with our hybrid continuum-atomistic model, and we show the model is in excellent agreement with fully-atomistic simulations of the nhTMEM16 lipid scramblase. We believe that the speed and accuracy of continuum-atomistic methodologies will make it possible to simulate large scale, slow biological processes, such as membrane morphological changes, that are currently beyond the scope of other computational approaches. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov