Initial Conformational Change Research Articles

Human T-cell receptor triggering requires inactivation of Lim kinase-1 by Slingshot-1 phosphatase

Actin dynamics control early T-cell receptor (TCR) signalling during T-cell activation. However, the precise regulation of initial actin rearrangements is not completely understood. Here, we have investigated the regulatory role of the phosphatase Slingshot-1 (SSH1) in this process. Our data show that SSH1 rapidly polarises to nascent cognate synaptic contacts and later relocalises to peripheral F-actin networks organised at the mature immunological synapse. Knockdown of SSH1 expression by CRISPR/Cas9-mediated genome editing or small interfering RNA reveal a regulatory role for SSH1 in CD3ε conformational change, allowing Nck binding and proper downstream signalling and immunological synapse organisation. TCR triggering induces SSH1-mediated activation of actin dynamics through a mechanism mediated by Limk-1 inactivation. These data suggest that during early TCR activation, SSH1 is required for rapid F-actin rearrangements that mediate initial conformational changes of the TCR, integrin organisation and proximal signalling events for proper synapse organisation. Therefore, the SSH1 and Limk-1 axis is a key regulatory element for full T cell activation.

Superstoichiometric Binding of the Anticancer Agent Titanocene Dichloride by Human Serum Transferrin and the Accompanying Lobe Closure.

Titanocene dichloride (TDC) is an anticancer agent that delivers Ti(IV) into each of the two Fe(III) binding sites of bilobal human serum transferrin (Tf). This protein has been implicated in the selective transport of Ti(IV) to cells. How Ti(IV) might be released from the Tf Fe(III) binding site has remained a question, and crystal structures have raised issues about lobe occupancy and lobe closure in Ti(IV)-loaded Tf, compared with the Fe(III)-loaded form. Here, inductively coupled plasma optical emission spectroscopy reveals that Tf can stabilize toward hydrolytic precipitation more than 2 equiv of Ti, implying superstoichiometric binding beyond the two Fe(III) binding sites. Further studies support the inability of TDC to induce a complete lobe closure of Tf. Fluorescence data for TDC binding at low equivalents of TDC support an initial protein conformational change and lobe closure upon Ti binding, whereas data at higher equivalents support an open lobe configuration. Spectroscopic titration reveals less intense protein-metal electronic transitions as TDC equivalents are increased. Denaturing urea-PAGE gels and small angle X-ray scattering studies support an open lobe conformation. The concentrations of bicarbonate used in some earlier studies are demonstrated here to cause a pH change over time, which may contribute to variation in the apparent molar absorptivity associated with Ti(IV) binding in the Fe binding site. Finally, Fe(III)-bound holo-Tf still stabilizes TDC toward hydrolytic precipitation, a finding that underscores the importance of the interactions of Tf and TDC outside the Fe(III) binding site and suggests possible new pathways of Ti introduction to cells.

Tracking Ultrafast Structural Dynamics in a Dual-Emission Anti-Kasha-Active Fluorophore Using Femtosecond Stimulated Raman Spectroscopy.

The anti-Kasha process provides the possibility of using high-energy excited states to develop novel applications. Our previous research (Nature communications, 2020, 11, 793) has demonstrated a dual-emission anti-Kasha-active fluorophore for bioimaging application, which exhibits near-infrared emissions from the S1 state and visible anti-Kasha emissions from the S2 state. Here, we applied tunable blue-side femtosecond stimulated Raman spectroscopy (FSRS) and transient absorption spectroscopy, assisted by quantum calculations, to reveal the anti-Kasha dual emission mechanism, in which the emergence of two fluorescing states is due to the retardation of internal conversion from the S2 state to the S1 state. It has been demonstrated that the facts of anti-Kasha high-energy emission are commonly attributed to a large energy gap between the two excited states, leading to a decrease in the internal conversion rate due to a poor Franck-Condon factor. In this study, analysis of the calculation and FSRS experimental results provide us further insight into the dual-emission anti-Kasha mechanism, where the observation of hydrogen out-of-plane Raman modes from FSRS suggested that, in addition to the energy-gap law, the initial photoinduced molecular conformational change plays a key role in influencing the rate of internal conversion.

Deciphering the role of trehalose in hindering antithrombin polymerization.

Serine protease inhibitors (serpins) family have a complex mechanism of inhibition that requires a large scale conformational change. Antithrombin (AT), a member of serpin superfamily serves as a key regulator of the blood coagulation cascade, deficiency of which leads to thrombosis. In recent years, a handful of studies have identified small compounds that retard serpin polymerization but abrogated the normal activity. Here, we screened small molecules to find potential leads that can reduce AT polymer formation. We identified simple sugar molecules that successfully blocked polymer formation without a significant loss of normal activity of AT under specific buffer and temperature conditions. Of these, trehalose proved to be most promising as it showed a marked decrease in the bead like polymeric structures of AT shown by electron microscopic analysis. A circular dichroism (CD) analysis indicated alteration in the secondary structure profile and an increased thermal stability of AT in the presence of trehalose. Guanidine hydrochloride (GdnHCl)-based unfolding studies of AT show the formation of a different intermediate in the presence of trehalose. A time-dependent fluorescence study using 1,1′-bi(4-anilino)naphthalene-5,5′-disulfonic acid (Bis-ANS) shows that trehalose affects the initial conformational change step in transition from native to polymer state through its binding to exposed hydrophobic residues on AT thus making AT less polymerogenic. In conclusion, trehalose holds promise by acting as an initial scaffold that can be modified to design similar compounds with polymer retarding propensity.

Twist and Degrade—Impact of Molecular Structure on the Photostability of Nonfullerene Acceptors and Their Photovoltaic Blends

AbstractNonfullerene acceptors (NFAs) dominate organic photovoltaic (OPV) research due to their promising efficiencies and stabilities. However, there is very little investigation into the molecular processes of degradation, which is critical to guiding design of novel NFAs for long‐lived, commercially viable OPVs. Here, the important role of molecular structure and conformation in NFA photostability in air is investigated by comparing structurally similar but conformationally different promising NFAs: planar O‐IDTBR and nonplanar O‐IDFBR. A three‐phase degradation process is identified: i) initial photoinduced conformational change (i.e., torsion about the core–benzothiadiazole dihedral), induced by noncovalent interactions with environmental molecules, ii) followed by photo‐oxidation and fragmentation, leading to chromophore bleaching and degradation product formation, and iii) finally complete chromophore bleaching. Initial conformational change is a critical prerequisite for further degradation, providing fundamental understanding of the relative stability of IDTBR and IDFBR, where the already twisted IDFBR is more prone to degradation. When blended with the donor polymer poly(3‐hexylthiophene), both NFAs exhibit improved photostability while the photostability of the polymer itself is significantly reduced by the more miscible twisted NFA. The findings elucidate the important role of NFA molecular structure in photostability of OPV systems, and provide vital insights into molecular design rules for intrinsically photostable NFAs.

“Effect of inhibiting protein conformational changes by introducing disulfide bonds in the transmembrane domains of the Hendra virus fusion protein”

Hendra virus (HeV) is a zoonotic pathogen that causes severe respiratory illness and encephalitis. However, no human vaccine or antiviral treatment is currently available. As a member of the Paramyxoviridae family, HeV consists of a non‐segmented negative‐sense RNA genome and is enclosed within a lipid bilayer which contains the surface glycoproteins known as the attachment protein (G) and the fusion protein (F). The G protein plays a role in target cell receptor binding, and the F protein promotes membrane fusion between the viral envelope and the target cell membrane to allow for viral entry and infection. Previous studies have shown that viral fusion proteins undergo conformational changes to drive fusion. For HeV, the F protein associates as a homotrimer, and research from our group suggests that the transmembrane domains (TMDs) stabilize the protein in its pre‐fusion state.1 Based on this research, we hypothesized that modulation of TMD interactions is critical for initiation and completion of the conformational changes that drive membrane fusion. To test this, three HeV F constructs (T483C/V484C, V484C/N485C, N485C/P486C) were generated with double cysteine mutations near the N‐terminal region of the TMD to study the effect of altered conformational flexibility in this region. Initially, the mutants were analyzed for intermolecular disulfide bond formation. Results showed that the mutants primarily associated in a trimeric form under non‐reducing conditions, in contrast to the wild type (WT) protein which was primarily monomeric, suggesting that the mutant TMDs were linked by disulfide bonds. Further experiments showed that all three mutants expressed in cells at levels similar to WT HeV F. However, cleavage of HeV F to its fusion‐active form was reduced for the three mutants, suggesting that the mutants are trafficked more slowly or undergo less proteolytic processing by the endosomal protease cathepsin L compared to WT HeV F. The mutants were then assessed for their ability to promote membrane fusion. Preliminary fusion studies showed that fusion was dramatically reduced for the mutants, suggesting that TMD dissociation is critical for initial conformational changes in HeV F. Future studies will further examine the conformational changes that can occur when dissociation of the TMDs is blocked by the introduction of disulfide bonds.Support or Funding InformationThe authors gratefully acknowledge funding from the NIH/NIAID, grant R01 AI051517.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The photodissociation of N,N-dimethylnitrosamine at 355 nm: The effect of excited-state conformational changes on product vector correlations

In a photodissociation experiment, the dynamics associated with creating reaction products with specific energies can be understood by a study of the product vector correlations. Upon excitation to the S1 state, N,N-dimethylnitrosamine (DMN) undergoes an excited-state geometry change from planar to pyramidal around the central N. The significant geometry change affects the vector correlations in the photoproducts. Using polarized lasers for 355 nm photodissociation of DMN and for NO photoproduct excitation in a velocity-mapped ion imaging apparatus reveals new vector correlation details among the parent transition dipole (μ), photofragment velocity (v), and photofragment angular momentum (j). The dissociation of DMN displays some μ-v correlation [β02(20)=-0.2], little μ-j correlation [β02(02)∼0], and, surprisingly, a v-j [β00(22)] correlation that depends on the NO lambda doublet probed. The results point to the importance of the initial excited-state conformational change and uncover the presence of two photolysis channels.

Cover Picture: Redesign of the Active Site of Sucrose Phosphorylase through a Clash‐Induced Cascade of Loop Shifts (ChemBioChem 1/2016)

The cover picture shows the active site and entrance channel of the Q345F variant of the sucrose phosphorylase from Bifidobacterium adolescentis (BaSP, PDB ID: 5C8B). Reshaping enzyme pockets paves the way for new enzyme activities. BaSP is a thoroughly studied enzyme and is frequently applied to glucosylate various acceptors. Unfortunately compounds of interest, for example bioactive plant polyphenols like flavonoids or resveratrol remain poor acceptors, presumably due to their spatial demands and polarity. We have created a new pocket for large aromatic acceptors through a cascade of loop shifts induced by a Gln345Phe exchange. The cover picture illustrates the initial conformational change of the cascade: sterically demanding Phe345 pushes the flexible Tyr344 out of the active site, thereby enabling the glucosylation of resveratrol. (Image created by Julian Görl.) More information can be found in the communication by C. Grimm, J. Seibel and M. Kraus on page 33 in Issue 1, 2016 (DOI: 10.1002/cbic.201500514).

Study of light-induced MscL gating by EPR spectroscopy.

A number of techniques developed to investigate protein structure and function depend on chemically modifying and/or labeling of proteins. However, in the case of homooligomeric proteins, the presence of multiple identical subunits obstructs the introduction of residue-specific labels to only one or several subunits, selectively. Here, in order to study the initial conformational changes of a homopentameric mechanosensitive ion channel during its gating, we developed a method for labeling a defined number of subunits of the channel with two different cysteine-specific compounds simultaneously. The first one is a light-sensitive channel activator that determines the degree of openness of the ion channel upon irradiation. The second one is a spin label, containing an unpaired electron, which allows following the resulting structural changes upon channel gating by electron paramagnetic resonance spectroscopy. With this method, we could open MscL into different sub-open states. As the number of light switches per channel increased, the intersubunit spin–spin interactions became less, indicating changes in intersubunit proximities and opening of the channel. The ability of controlled activation of MscL into different open states with a noninvasive trigger and following the resulting conformational changes by spectroscopy will pave the way for detailed spectroscopic studies in the area of mechanosensation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00249-015-1063-4) contains supplementary material, which is available to authorized users.

Reaction dynamics of the UV-B photosensor UVR8.

UVR8 is a recently discovered ultraviolet-B (UV-B) photoreceptor protein identified in plants and algae. In the dark state, UVR8 exists as a homodimer, whereas UV-B irradiation induces UVR8 monomerization and initiation of signaling. Although the biological functions of UVR8 have been studied, the fundamental reaction mechanism and associated kinetics have not yet been fully elucidated. Here, we used the transient grating method to determine the reaction dynamics of UVR8 monomerization based on its diffusion coefficient. We found that the UVR8 photodissociation reaction proceeds in three stages: (i) photoexcitation of cross-dimer tryptophan (Trp) pyramids; (ii) an initial conformational change with a time constant of 50 ms; and (iii) dimer dissociation with a time constant of 200 ms. We identified W285 as the key Trp residue responsible for initiating this photoreaction. Although the C-terminus of UVR8 is essential for biological interactions and signaling via downstream components such as COP1, no obvious differences were detected between the photoreactions of wild-type UVR8 (amino acids 1-440) and a mutant lacking the C-terminus (amino acids 1-383). This similarity indicates that the conformational change associated with stage ii cannot primarily be attributed to this region. A UV-B-driven conformational change with a time constant of 50 ms was also detected in the monomeric mutants of UVR8. Dimer recovery following monomerization, as measured by circular dichroism spectroscopy, was decreased under oxygen-purged conditions, suggesting that redox reactivity is a key factor contributing to the UVR8 oligomeric state.

Detailed molecular dynamics simulations of human transferrin provide insights into iron release dynamics at serum and endosomal pH.

Human serum transferrin (hTf) transports ferric ions in the blood stream and delivers them to cells via receptor-mediated endocytosis. hTf is folded into two homologous lobes; we utilize three of the available crystal structures delineating large conformational changes involved in iron binding/dissociation. We address the problems of whether the release process follows the same trend at serum (~7.4) and endosomal (~5.6) pH, and if there is communication between the lobes. In the absence of the transferrin receptor, we study the dynamics of the full structure as well as the separate lobes in different closed, partially open, and open conformations under neutral and endosomal pH conditions. Results corroborate those experimental observations underscoring the distinguishing effect of pH on the dynamics of hTf. Furthermore, in a total of 2 μs molecular dynamics simulations, residue fluctuations elucidate the cross talk between the lobes correlated by the peptide linker bridging them at serum pH, while their correlations are lost under endosomal conditions. At serum pH, interplay between relative mobility of the lobes is correlated with iron release rates, rendering the initial conformational change an important contributor to the dynamics under these conditions. Interestingly, C-lobe opening lags behind that of the N-lobe as long as there is at least one iron bound, making the more stable C-lobe an attractive target for recognition by receptors. At endosomal pH, both lobes readily open, making irons available for delivery.

Moving along the Free Energy Landscape of Membrane Insertion of the Diphtheria Toxin Translocation Domain

The pH-triggered membrane insertion of the translocation (T) domain is critical for the entry of the diphtheria toxin into the target cell. Previously we characterized the kinetic pathway of membrane insertion of the T domain, which consists of a sequence of conformational changes that convert a water-soluble state into a transmembrane state. Here we utilize various thermodynamic approaches to determine the changes in the Gibbs free energy associated with these conformational changes. The initial conformational change, which occurs in solution, was studied by thermal and chemical denaturation using differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. We found that acidification of solution, which results in the formation of the membrane-competent form, reduces the thermodynamic stability of the T-domain by about 3-5 kcal/mol, depending on the experimental conditions. For thermodynamic studies of membrane insertion we applied a novel approach that combines fluorescence correlation spectroscopy with the use of fluorinated surfactants as chemical chaperones. We estimated that the free energy values for the transition from a membrane-competent state in solution to the interfacial intermediate, and to the final transmembrane state are about −8.2 ± 0.2 kcal/mol and −12.0 ± 0.2 kcal/mol, respectively. The free energy barrier between the two states is modulated by the presence of anionic lipids. We summarize our findings in a proposed free energy landscape for the refolding and bilayer insertion of the T domain. Supported by NIH GM-069783 (A.S.L.), and Fulbright-CONICYT and BRTP (M.V.U.)

Dehydration-induced initial conformational change of hydrated proteins detected by the changes in vibrational circular dichroism activity.

Conformational changes of hydrated proteins induced by gradual dehydration were monitored by vibrational circular dichroism (VCD) spectroscopy. In myoglobin and casein, representative α-helix-rich and random-coil proteins, respectively, an increase in left-handed optical activity in the amide I band was detected at the initial stage of dehydration, followed by an increase in opposite right-handed activity in both the amide I and II bands with further dehydration. Because the second step was observed with an increase in the turbidity of the proteins, it can be attributed to their aggregation. In contrast, because the increase in left-handed optical activity is induced by the conformational change of the proteins and is followed by the aggregation, it may derive from the increase in the regularity of the local structure in individual myoglobin or casein that triggers the aggregation.

Molecular dynamics simulations reveal initial structural and dynamic features for the A2AR as a result of ligand binding

G-protein-coupled receptors (GPCRs) are membrane proteins that have a wide variety of physiological roles. Adenosine receptors belong to the GPCR family. Adenosine receptors are implicated in many physiological disorders, such as Parkinson's disease, Huntington's disease, inflammatory and immune's disease and many others. Interestingly, crystal structures of the active and inactive conformations of the A2-subtype adenosine receptor (A2AR) have been solved. These two structures could be used to get insights about the conformational changes that occur during the process of activation/inactivation processes of this receptor. Therefore, two ligand-free simulations of the native active (PDB code: 3QAK) and inactive (PDB code: 3EML) conformations of the A2AR and two halo-simulations were carried out to observe the initial conformational changes induced by coupling adenosine to the inactive conformation and caffeine to the active conformation. Furthermore, we constructed an A2AR model that contained four thermostabilising mutations, L48A, T65A, Q89A and A54L, which had previously been determined to stabilise the bound conformation of the agonist, and we ran molecular dynamics simulations of this mutant to investigate how these point mutations might affect the inactive conformation of this receptor. This study provides insights about the initial structural and dynamic features that occur as a result of the binding of caffeine and adenosine in the active and inactive A2AR structures, respectively, as well as the introduction of some mutations on the inactive structure of the A2AR. Moreover, we provide useful and detailed information regarding structural features such as toggle switch and ionic lock during the activation/inactivation processes of this receptor.

Heparin increases the infectivity of Human Papillomavirus Type 16 independent of cell surface proteoglycans and induces L1 epitope exposure

Human Papillomaviruses (HPVs) are the etiological agents of cervical cancer, and HPV-16 is the most prevalent type. Several HPVs require heparan sulfate proteoglycans (HSPGs) for cell binding. Here, we analyse the phenomenon that preincubation of HPV-16 with increasing concentrations of heparin results in partial restoration rather than more efficient inhibition of infection. While corroborating that the HSPGs are cell-binding receptors for HPV-16, heparin-preincubated virus bound to the extracellular matrix (ECM) via laminin-332. Furthermore, the interaction of virions with heparin, a representative of the highly sulfated S-domains of heparan sulfate (HS) chains of HSPGs, allowed HPV-16 infection in the absence of cell surface HSPGs. Therefore, we concluded that specific glycan moieties but not specific HSPG protein backbones are required for infection. The increased binding of an epitope-specific antibody to the viral capsid after heparin binding suggested that initial conformational changes in the HPV-16 virion occur during infection by interaction with'heparin-like' domains of cellular HSPGs. We propose that HS sequences with specific sulfation patterns are required to facilitate HPV-16 infection.

SnapShot: Viral and Eukaryotic Protein Fusogens

Viral Fusion Effectors Viral fusogens are anchored in the viral membrane, and the initial fusogenic conformational change allows them to insert into the cell membrane. This transition is triggered by interactions of the virus particle with the host cell—either binding a cell surface receptor or protons in the acidic endosomal environment. During the fusogenic conformational change, the fusion protein initially adopts an intermediate extended conformation in which a “fusion peptide” projects away from the virus particle and inserts into the target membrane. The fusion protein subsequently collapses into a “hairpin” conformation in which the fusion peptide becomes juxtaposed to the viral transmembrane (TM) segment, thereby juxtaposing the two membranes. The structures of the postfusion hairpin conformation for a number of viruses are presented, highlighting common structural features of the observed hairpins. The overall process of virus-cell fusion and the accompanying conformational changes of the effector proteins have been reviewed, see for example Harrison (2008). The viral fusion proteins analyzed so far fall into three structural classes in which the postfusion form is a trimer of hairpins with different architectures. The predicted location of the fused membrane is outlined above the trimers. The class I motif is characterized by a 7 residue periodicity of nonpolar amino acids (i.e.,“heptad repeats”) that give rise to a central parallel trimeric a-helical coiled coil along the long axis of a rod-shaped molecule (blue in the ribbon diagrams). The N and C termini (labeled in blue and cyan) pack against each other at the membrane proximal end. A “stutter,” a region where the coiled helix unwinds and alters the nonpolar repeat pattern (in red), permits the structural alignment of the various hairpin trimers (Igonet et al., 2011). The heptad repeats are also interrupted by hydrophilic layers (indicated in magenta, with horizontal arrows). Class II proteins are folded essentially as b sheets with no long coiled a helix, and the trimer of hairpins is formed by the parallel interactions of domains I and II (red and yellow). The trimer is further clamped in place by the lateral interactions of domain III (in blue), which stabilizes the hairpin conformation. A stem region (only partially present in the available structures) connects to the TM segment. The fusion peptide is internal, situated in the loop between two b strands, and is therefore termed “fusion loop” (FL). Class III proteins appear to be intermediate between classes I and II: they have a central parallel coiled coil (blue), but they have an elongated, b-sheet-rich fusion domain N terminal to it (yellow) instead of a fusion peptide as in class I. Although this fusion domain is superficially similar to its counterpart in class II proteins, the topological b-strand organization is unrelated, suggesting convergent rather than divergent evolution. The central parallel coiled coil also has a stutter, allowing a close alignment to the class I fusion proteins. The fusion domain of class III proteins has two fusion loops labeled FL1 and FL2. Although there is no amino acid sequence conservation across virus families, the structures show clear homology within classes II and III. However, the simple heptad repeat pattern of class I fusogens is in several cases not sufficient to distinguish between divergent or convergent evolution and therefore, only for a subset of families displayed in the figure do the fusion proteins appear as clearly homologous. Fusion of Intracellular Vesicles A variety of different proteins control the fusion of intracellular vesicles, but the actual fusion effectors are believed to be the SNARE proteins, as reviewed by Sudhof and Rothman (2009). The prefusion form of the SNAREs appears to be largely unstructured, and the postfusion form consists of a hetero-oligomeric complex formed by SNARE subunits that were anchored in the two opposing membranes before their fusion. The SNAREs in their postfusion conformation share with viral proteins of classes I and III an elongated, a-helical, parallel oligomeric coiled-coil organization in which the C-terminal TM segments are juxtaposed at the same end, making a stable protein rod termed “SNAREpin” by analogy to the viral hairpin. The SNAREpin organization differs from the viral proteins because it is a four-stranded coiled coil in which the amino acid sequence of each a helix is different. At the center, there is a hydrophilic, or “ionic,” layer consisting of a central arginine (R) and three glutamine (Q) residues that hydrogen bond the guanidinium group of the arginine. This feature has allowed the classification of the SNAREs into four different categories—R-SNAREs and Qa-, Qb-, and Qc-SNAREs—with one representative of each being necessary for formation of a functional SNAREpin, as displayed in the figure.

The Transition State of Coupled Folding and Binding for a Flexible β-Finger

Flexible and fully disordered protein regions that fold upon binding mediate numerous protein–protein interactions. However, little is known about their mechanism of interaction. One such coupled folding and binding occurs when a flexible region of neuronal nitric oxide synthase adopts a β-finger structure upon binding to its protein ligand, a PDZ [PSD-95 (postsynaptic density protein-95)/Discs large/ZO-1] domain from PSD-95. We have analyzed this binding reaction by protein engineering combined with kinetic experiments. Mutational destabilization of the β-finger changed mainly the dissociation rate constant of the proteins and, to a lesser extent, the association rate constant. Thus, mutation affected late events in the coupled folding and binding reaction. Our results therefore suggest that the native binding interactions of the β-finger are not present in the rate-limiting transition state for binding but form on the downhill side in a cooperative manner. However, by mutation, we could destabilize the β-finger further and change the rate-limiting step such that an initial conformational change becomes rate limiting. This switch in rate-limiting step shows that multistep binding mechanisms are likely to be found among flexible and intrinsically disordered regions of proteins.

Activation of Ms Channel by LPC: A Trigger for MscL Gating in the Absence of Applied Tension

Mechanosensitive Channel of Large Conductance (MscL) allows bacteria to respond to osmotic stress in the environment. It senses the increase in the lateral pressure in the membrane due to sudden hypo-osmotic shock and acts as a safety valve. MscL has one of the largest pores in nature; in its open state it allows the passage of ions and small molecules upto 6.5 kDa. MscL has been used in this study as an externally controlled valve i.e. the opening of the channel is controlled by external stimuli.Several techniques like patch clamp, EPR spectroscopy has been applied towards elucidating the gating mechanism of MscL. EPR is effective in tracking the initial conformational changes that the protein may undergo during gating. The main challenge in using spectroscopy is that, unlike patch clamp technique, tension cannot be applied directly for opening the channel. L-α-lysophosphatidylcholine, a reported activator of MscL was studied in this work to trigger opening of the channel in a controlled way. In our work we provide evidence that LPC mimics tension in opening the channel. Our findings also clearly show that LPC can be used for phenotypic characterization of MscL mutants, in a much simpler experiment than patch clamp. A clear differentiation in activity between GOF, LOF and Wt Ec MscL is observed at 4 μM LPC. In conclusion, we characterized an activator with which the mechanism of channel gating can be studied in a controlled way.

Real Time Observation of Ultrafast Peptide Conformational Dynamics: Molecular Dynamics Simulation vs Infrared Experiment

Employing nonequilibrium molecular dynamics (MD) simulations and transient infrared (IR) spectroscopy, a joint theoretical/experimental study on a water-soluble photoswitchable octapeptide designed by Renner et al. [Biopolymers 2002, 63, 382] is presented. The simulations predict the cooling of the hot photoproducts on a time scale of 7 ps and complex conformational rearrangements ranging from a few picoseconds to several nanoseconds. The experiments yield a dominant fast relaxation time of 5 ps, which is identified as the cooling time of the peptide in water and also accounts for initial conformational changes of the system. Moreover, a weaker component of 300 ps is found, which reflects the overall conformational relaxation of the system. The virtues and the limitations of the joint MD/IR approach to describe biomolecular conformational rearrangements are discussed.

Simple, Automated, High Resolution Mass Spectrometry Method to Determine the Disulfide Bond and Glycosylation Patterns of a Complex Protein: SUBGROUP A AVIAN SARCOMA AND LEUKOSIS VIRUS ENVELOPE GLYCOPROTEIN

Enveloped viruses must fuse the viral and cellular membranes to enter the cell. Understanding how viral fusion proteins mediate entry will provide valuable information for antiviral intervention to combat associated disease. The avian sarcoma and leukosis virus envelope glycoproteins, trimers composed of surface (SU) and transmembrane heterodimers, break the fusion process into several steps. First, interactions between SU and a cell surface receptor at neutral pH trigger an initial conformational change in the viral glycoprotein trimer followed by exposure to low pH enabling additional conformational changes to complete the fusion of the viral and cellular membranes. Here, we describe the structural characterization of the extracellular region of the subgroup A avian sarcoma and leukosis viruses envelope glycoproteins, SUATM129 produced in chicken DF-1 cells. We developed a simple, automated method for acquiring high resolution mass spectrometry data using electron capture dissociation conditions that preferentially cleave the disulfide bond more readily than the peptide backbone amide bonds that enabled the identification of disulfide-linked peptides. Seven of nine disulfide bonds were definitively assigned; the remaining two bonds were assigned to an adjacent pair of cysteine residues. The first cysteine of surface and the last cysteine of the transmembrane form a disulfide bond linking the heterodimer. The surface glycoprotein contains a free cysteine at residue 38 previously reported to be critical for virus entry. Eleven of 13 possible SUATM129 N-linked glycosylation sites were modified with carbohydrate. This study demonstrates the utility of this simple yet powerful method for assigning disulfide bonds in a complex glycoprotein.