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In Situ Solid-State Nmr Spectroscopy of APP Transmembrane Domain (TM) Structure and Dimerization in Native E. Coli Membranes
Wednesday, February 15, 2017 we showed that allosteric communication between the antagonist binding site and the IC interface stabilizes the inactive state, and disruption of this allosteric communication due to agonist binding leads to activation. Furthermore, the fundamental framework and mechanism of allosteric communication are conserved among multiple GPCRs, as is evident from the conserved allosteric hubs (residues that mediate multiple allosteric pathways) in these receptors. Mutating the allosteric hubs either suppress activation or impart constitutive activity, suggesting the key role of these residues in GPCR function. We have shown that, besides stabilizing GPCR functional states, allosteric communication is also responsible for transitions among functional states. Analysis of the deactivation dynamics of two class A GPCRs (b2 adrenergic receptor and neurotensin receptor 1) shows that several allosteric hubs in the middle of the transmembrane domain undergo concerted dihedral changes during the transition from the active to the inactive state. These observations provide valuable insights into the complex mechanism of GPCR activation and membrane protein function in general. 2455-Pos Board B62 Deciphering General Characteristics of Residues Constituting Allosteric Communication Paths Girik Malik, Anirban Banerji, Andrzej Kloczkowski. Battelle Center for Mathematical Medicine, Nationwide Children’s Hopital, Columbus, OH, USA. There has been great interest in studying proteins’ dynamics and structural changes involved in function to gain insights into the machinery of proteins. Due to difficulty in retaining atomic details in mode decomposition of large system dynamics, there have been significant computational challenges, which make the study of large system dynamics very complex. Considering all the PDB annotated proteins from the Allosteric Database (ASD) [1,2] belonging to four different classes (kinases, nuclear receptors, peptidases, transcription factors), this work has attempted to decipher certain consistent patterns present in the residues constituting the allosteric communication sub-system (ACSS). While the thermal fluctuations of hydrophobic residues in ACSSs were found to be significantly higher than those present in the non-ACSS part of the same proteins, thermal fluctuations recorded for the polar residues showed the opposite trend. While the basic residues and hydroxyl residues were found to be slightly more predominant than the acidic residues and amide residues in ACSSs, hydrophobic residues were found extremely frequently in kinase ACSSs. Despite having different sequences and different lengths of ACSS, they were found to be structurally quite similar to each other, suggesting a preferred structural template for communication. ACSS structures recorded low RMSD and high Akaike Information Criterion (AIC) scores among themselves. While the ACSS networks for all the groups of allosteric proteins recorded low degree centrality and closeness centrality, the betweenness centrality magnitudes revealed non-uniform behavior. Though cliques and communities could be identified within the ACSS, maximalcommon-subgraph considering all the ACSS could not be generated, primarily due to the diversity in the dataset. Barring one particular case, the entire ACSS for any class of allosteric proteins did not demonstrate ‘‘small world’’ behavior, except for a few sub-graphs. Such a report can be expected to benefit the protein engineering community, those who attempt to decipher the general mechanism of allostery, and in general, long-distance communication within protein structures, both from knowing the topological invariants of communication paths and from knowing the biophysical-biochemical-and-structural patterns therein. [1] Huang Z, Zhu L, Cao Y, et al. ASD: a comprehensive database of allosteric proteins and modulators. Acids Res. 2011; 39 (Database issue):D663-D669. [2] Huang Z, Mou L, Shen Q, et al. ASD v2.0: updated content and novel features focusing on allosteric regulation. Nucleic Acids Res. 2014; 42 (Database issue):D510-D516. 2456-Pos Board B63 Reweighting the Apo to the Holo Ensemble Chen Li. Chemisty, IIT, Chicago, IL, USA. Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site. Simulations can enable the discovery of allosteric binding sites and elucidate the mechanism of allostery, providing a foundation for applications including rational drug design and protein engineering. However, performing a separate simulation for every ligand can be computationally expensive. Here we demonstrate a proof of principle that conformations sampled from the apo state can be reweighted according to the binding potential of mean force (BPMF) to predict ensemble averages in the holo state. Using BPMFs for the binding of AMP and
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ADP to adenylate kinase, an enzyme that plays an important role in the process of energy homeostasis, we construct a hinge angle distribution consistent with alchemical binding free energy calculations. 2457-Pos Board B64 Opposing Intermolecular Tuning of Ca2D Affinity for Calmodulin by Target Peptides Margaret S. Cheung, Pengzhi Zhang, Swarnendu Tripathi. University of Houston, Houston, TX, USA. We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca2þ) by integrating coarse-grained models and all-atomistic simulations with non-equilibrium physics. We focused on binding between CaM and two specific targets, Ca2þ/CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaMdependent Ca2þ signaling pathways in neurons. It was shown experimentally that Ca2þ/CaM binds to the CaMKII peptide with higher affinity than the Ng peptide. The binding of CaMKII peptide to CaM in return increases the Ca2þ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide that binds to Ca2þ-free CaM or Ca2þ/CaM with similar binding affinity. Unlike CaM-CaMKII peptide that renders solved crystal structures, the structural description of CaM-Ng peptide is unknown due to low binding affinity, therefore, we computationally generated an ensemble of CaM-Ng peptide structures by matching the changes in the chemical shifts of CaM upon Ng peptide binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca2þ affinity for CaM with and without binding targets in atomistic models using Jarzynski’s equality. We discovered the molecular underpinnings of lowered affinity of Ca2þ for CaM in the presence of Ng by showing that the N-terminal acidic region of Ng peptide pries open the b-sheet structure between the Ca2þ binding loops particularly at C-domain of CaM, enabling Ca2þ release. In contrast, CaMKII increases Ca2þ affinity for the C-domain of CaM by stabilizing the two Ca2þ binding loops.
Membrane Protein Structures II 2458-Pos Board B65 Comparing and Contrasting Fluorotryptophan Substitutions for 19F Membrane Protein NMR Spectroscopy Calem Kenward, Kyungsoo Shin, Muzaddid Sarker, Carley Bekkers, Jan K. Rainey. Dalhousie University, Halifax, NS, Canada. The high gyromagnetic ratio and broad chemical shift range of 19F make it sensitive and valuable as a probe for studying protein conformation, dynamics and intermolecular interactions by NMR spectroscopy. In this work, we are comparing strategies for 19F NMR characterization of membrane proteins with the goal of developing a straightforward, robust, and versatile 19F-labeling scheme. One popular and frequently used approach is the incorporation of 19Fsubstituted aromatic amino acids. Using a recently introduced method showing efficient and cost-effective 5-fluorotryptophan protein labeling through addition of 5-fluoroindole to M9 minimal medium (Crowley et al. (2012) Chem Commun 48: 10681), we have extended this approach for incorporation of 19 F labels at the 4-, 5-, 6-, and 7-positions. Two different fragments of the apelin receptor (AR), a class A G-protein coupled receptor (GPCR), were employed: AR55 (residues 1-55 including the first transmembrane (TM) segment) and AR TM1-3 (residues 1-137 including the first 3 TM segments). We have previously characterized both of these fragments previously using heteronuclear NMR methods in a variety of membrane-mimetic conditions. AR55 has two Trp residues, each residing close to the headgroup region of micelles. AR TM1-3 has two additional Trp residues, one of which is predicted to fall in TM2, the other in extracellular loop 1. The effectiveness of the four fluorotryptophan substitutions is compared both in terms of chemical shift dispersion and in terms of relaxation behavior. These are compared and contrasted both for the topological location of a given tryptophan (assigned by mutagenesis) and for micellar environments with different headgroup chemistry (i.e., zwitterionic vs. anionic headgroups). On the basis of the observed differences, strategies are suggested for the most effective fluorotryptophan incorporation and application for 19F NMR studies of membrane protein topology, structural change, dynamics, and ligand binding. 2459-Pos Board B66 In Situ Solid-State Nmr Spectroscopy of APP Transmembrane Domain (TM) Structure and Dimerization in Native E. Coli Membranes Xiaoyan Ding1, Riqiang Fu2, Fang Tian1. 1 Penn State College of Medicine, Hershey, PA, USA, 2National High Magnetic Field Laboratory, Tallahassee, FL, USA. Cleavage of the amyloid precursor protein (APP) by a-, b- and g-secretases leads to generation of amyloid peptide (Ab), which is the primary constituent
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of Alzheimer’s disease (AD) plaques in the brain. Three consecutive GXXXG motifs and one GXXXA motif on the transmembrane domain (TM) of APP have been reported to be one or two important sites for mediating APP homodimerization in different experimental conditions, the difference are likely due to the membrane mimetic. We and several other groups recently demonstrated the feasibility to directly characterize recombinant proteins in native Escherichia coli (E. coli) membranes using magic-angle spinning (MAS) solid-state nuclear magnetic resonance (ssNMR)(2, 3). Here, we will present several strategies to further improve spectral sensitivity and resolution, and suppress background signals of E. coli proteins and lipids, including preparation of E. coli inner membranes, reverse 13C- and 15N-isotope labeling, and selective signal filtering with the frequency-selective REDOR pulse sequence. These improvements have allowed us to characterize the structure and dimerization of APP TM and its variants in a native membrane environment. 2460-Pos Board B67 Structural and Dynamical Basis of Protein Kinase C Alpha Regulation by the C-Terminal Tail Yuan Yang1, Julia A. Callender2, Alexandra C. Newton2, Tatyana I. Igumenova1. 1 Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA, 2Department of Pharmacology, University of California at San Diego, La Jolla, CA, USA. Protein Kinase C (PKC) isoenzymes are dynamic multi-modular proteins that regulate signal transduction processes at the membrane surface. When activated by second messengers, such as Ca2þ and diacylglycerol, PKC undergoes a drastic conformational transition from the inactive cytosolic state to the activated membrane-bound state. The structure of either state of PKC is not known. Using NMR, we demonstrate that the isolated Ca2þ-sensing lipid-binding C2 domain of PKCa interacts with the C-terminal tail of the kinase. We determined the structure of the complex between the C2 domain and the peptide corresponding to the phosphorylated hydrophobic motif of the C-terminal tail. The structure shows that the hydrophobic motif binds to the conserved lysine-rich cluster region of C2. The interface is stabilized by electrostatic interactions and stacking of the aromatic sidechains. We validated our structural model by mutating the interacting residues in full-length PKC, and characterizing these variants using in vitro FRET experiments and FRET-detected membrane translocation experiments in live cells. In addition, NMR-detected binding studies revealed that the hydrophobic motif and Ca2þ synergistically enhance each other’s affinities to C2. We propose a model where the C-terminal tail plays a dual role in PKC regulation: auto-inhibitory, through its interaction with the lysine-rich cluster of the C2 domain; and activating, through sensitization of PKC to intracellular Ca2þ oscillations. Support: Welch Foundation grant A-1784 (TII), NSF CAREER award CHE-1151435 (TII), NIH GM108998 (TII), and NIH GM43154 (ACN). 2461-Pos Board B68 Site-Directed Spin Labeling EPR Spectroscopy of the Cytoplasmic Tail of Influenza a M2 Alice L. Herneisen, Grace Kim, Kathleen P. Howard. Swarthmore College, Swarthmore, PA, USA. The M2 protein is a 97 residue homotetrameric, multifunctional ion channel that plays critical roles during the influenza infection cycle. While a variety of high-resolution biophysical techniques have been used to characterize the transmembrane domain (residues 22-46) and the juxtamembrane C-terminal region (46-62), less is known about the conformation and dynamics of the remaining residues of the C-terminal cytoplasmic tail. Here, we use site-directed spin labeling electron paramagnetic spectroscopy (SDSL-EPR) experiments to probe the secondary structure and membrane topology of cytoplasmic tail residues 60-80 when the protein is reconstituted into lipid bilayers. Cholesterol is essential for the role the C-terminal domain of the M2 protein plays in viral budding. SDSL-EPR data is collected in both in the presence and absence of cholesterol. 2462-Pos Board B69 Polarity and Charge as Determinants for Translocase Requirement for Membrane Protein Insertion Balasubramani Hariharan1, Raunak Soman2, Ross E. Dalbey3. 1 Biophysics Graduate program, The Ohio State University, Columbus, OH, USA, 2OSBP, The Ohio State University, Columbus, OH, USA, 3The Ohio State University, Columbus, OH, USA. Membrane protein biogenesis in bacteria is a well-studied but poorly understood area in the field of membrane proteins. In bacteria, the Sec translocon and the YidC insertase promote the membrane insertion process. Both proteins are essential, and universally conserved, and together insert about 95% of the proteins in E.coli. Membrane insertion of SecYEG-
dependent proteins likely occurs at the lateral gate of SecY, the channelforming subunit of the bacterial Sec translocon. On the other hand, YidC catalyzes insertion by possessing a hydrophilic groove, which can accommodate the hydrophilic region of the substrate that inserts across the membrane. Recently, Soman et al (2014) have reported that increasing the charge of the translocated region of procoat can route substrates from the YidC pathway into the Sec pathway. Here, we have further tested this polarity/charge hypothesis by showing that the major coat protein of bacteriophage M13 can become increasingly YidC/Sec dependent by making the periplasmic loop highly polar in the absence of charged residues. We also show that adding hydrophobic amino acids to highly polar or charged loop can decrease the Secdependence of the otherwise strictly Sec-dependent substrates. Additionally we demonstrate that increasing the driving force of insertion by adding four leucyl residues to the transmembrane segment leads to translocation of a highly charged region that was not inserted by native TM segment. Finally, we show that the length of procoat loop is a determinant for Sec dependent insertion. The combined results support the polarity/charge hypothesis and is consistent with the notion that membrane insertion occurs at the interface of SecYEG and YidC. 2463-Pos Board B70 Bip Binding Affects Integration of Transmembrane Domains Mirjam Zimmermann1,2, Marco Janoschke2, Martin Spiess2. 1 Molecular and Membrane Biophysics, Institute of Biophysics, Linz, Austria, 2 Biozentrum, Basel, Switzerland. The Sec61 translocon mediates translocation of proteins into the endoplasmic reticulum (ER) and enables integration of hydrophobic (H) segments into the lipid bilayer via its lateral gate. The luminal chaperone BiP (Kar2 in yeast) has been shown to act as a molecular ratchet, recruited to the emerging polypeptide by the J-domain of Sec63, and to be involved in post- and cotranslational translocation. In this study, we address the question whether BiP binding to the nascent chain affects the process of membrane integration vs. translocation, i.e. whether integration is governed by thermodynamic equilibrium between pore and membrane. To determine the hydrophobicity threshold of membrane integration, a series of model proteins based on the sequence of dipeptidylaminopeptidase B (DPAPB) with oligo-alanine H-segments containing increasing numbers of leucines. The translocated loop preceding the H-segments was replaced by a sequence of similar length containing multiple copies of segments known to bind BiP with high affinity or not to interact with BiP. While H-segments preceded by the DPAPB sequence required ~3.7 Leu for 50% integration, this threshold was reduced to ~2.1 Leu with the BiP-binding sequence and increased to ~5.2 Leu with the non-binding sequence. These results show that membrane integration of an H-segment is influenced by the already translocated sequence preceding it and suggest that BiP binding promotes membrane integration. We observed changes of the apparent free energy in integration between 1 to 3 kcal/mol. We propose a model in which Brownian motion of the translocated loop with bound chaperones preferentially promotes release of the H-segment into the lipid bilayer. The work was supported by grant W0125 of the Austrian science fund (FWF). 2464-Pos Board B71 Elucidating the Uncoupling of ATP Hydrolysis and Ca2D Transport in SERCA by Sarcolipin Erin Birdsall1, Alysha Dicke1, Gianluigi Veglia1,2. 1 BMBB, University of Minnesota, Minneapolis, MN, USA, 2Chemistry, University of Minnesota, Minneapolis, MN, USA. The activity of the sarco(endo)plasmic reticulum Ca2þ -ATPase (SERCA), which plays a key role in muscle contraction and relaxation, is regulated by sarcolipin (SLN), a 31-residue transmembrane peptide. Sarcolipin has been shown to uncouple ATP hydrolysis and Ca2þ transport in SERCA and is largely found in skeletal muscle and the atria of the heart. The interaction between SLN and SERCA is thought to play an important role in energy metabolism. SLN is composed of a transmembrane helix flanked by two short, unstructured Nand C-termini. The N-terminus is hypothesized to play a role in the uncoupling of SERCA, while the role of the C-terminus has been shown to be largely responsible for SERCA inhibition. In this study, we made two constructs of SLN, with either the C- or N-terminus deleted. We then used coupledenzyme activity assays to measure the effect of the SLN constructs on SERCA ATPase activity and isothermal titration calorimetry (ITC) to monitor the change in heat release due to ATP hydrolysis. We found through these methods that the deletion of the termini led effected the uncoupling function in SLN. The results of this study, in combination with forthcoming solid-state NMR studies, will help to establish the mechanism by which SLN regulates SERCA activity.
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ADP to adenylate kinase, an enzyme that plays an important role in the process of energy homeostasis, we construct a hinge angle distribution consistent with alchemical binding free energy calculations. 2457-Pos Board B64 Opposing Intermolecular Tuning of Ca2D Affinity for Calmodulin by Target Peptides Margaret S. Cheung, Pengzhi Zhang, Swarnendu Tripathi. University of Houston, Houston, TX, USA. We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca2þ) by integrating coarse-grained models and all-atomistic simulations with non-equilibrium physics. We focused on binding between CaM and two specific targets, Ca2þ/CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaMdependent Ca2þ signaling pathways in neurons. It was shown experimentally that Ca2þ/CaM binds to the CaMKII peptide with higher affinity than the Ng peptide. The binding of CaMKII peptide to CaM in return increases the Ca2þ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide that binds to Ca2þ-free CaM or Ca2þ/CaM with similar binding affinity. Unlike CaM-CaMKII peptide that renders solved crystal structures, the structural description of CaM-Ng peptide is unknown due to low binding affinity, therefore, we computationally generated an ensemble of CaM-Ng peptide structures by matching the changes in the chemical shifts of CaM upon Ng peptide binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca2þ affinity for CaM with and without binding targets in atomistic models using Jarzynski’s equality. We discovered the molecular underpinnings of lowered affinity of Ca2þ for CaM in the presence of Ng by showing that the N-terminal acidic region of Ng peptide pries open the b-sheet structure between the Ca2þ binding loops particularly at C-domain of CaM, enabling Ca2þ release. In contrast, CaMKII increases Ca2þ affinity for the C-domain of CaM by stabilizing the two Ca2þ binding loops.
Membrane Protein Structures II 2458-Pos Board B65 Comparing and Contrasting Fluorotryptophan Substitutions for 19F Membrane Protein NMR Spectroscopy Calem Kenward, Kyungsoo Shin, Muzaddid Sarker, Carley Bekkers, Jan K. Rainey. Dalhousie University, Halifax, NS, Canada. The high gyromagnetic ratio and broad chemical shift range of 19F make it sensitive and valuable as a probe for studying protein conformation, dynamics and intermolecular interactions by NMR spectroscopy. In this work, we are comparing strategies for 19F NMR characterization of membrane proteins with the goal of developing a straightforward, robust, and versatile 19F-labeling scheme. One popular and frequently used approach is the incorporation of 19Fsubstituted aromatic amino acids. Using a recently introduced method showing efficient and cost-effective 5-fluorotryptophan protein labeling through addition of 5-fluoroindole to M9 minimal medium (Crowley et al. (2012) Chem Commun 48: 10681), we have extended this approach for incorporation of 19 F labels at the 4-, 5-, 6-, and 7-positions. Two different fragments of the apelin receptor (AR), a class A G-protein coupled receptor (GPCR), were employed: AR55 (residues 1-55 including the first transmembrane (TM) segment) and AR TM1-3 (residues 1-137 including the first 3 TM segments). We have previously characterized both of these fragments previously using heteronuclear NMR methods in a variety of membrane-mimetic conditions. AR55 has two Trp residues, each residing close to the headgroup region of micelles. AR TM1-3 has two additional Trp residues, one of which is predicted to fall in TM2, the other in extracellular loop 1. The effectiveness of the four fluorotryptophan substitutions is compared both in terms of chemical shift dispersion and in terms of relaxation behavior. These are compared and contrasted both for the topological location of a given tryptophan (assigned by mutagenesis) and for micellar environments with different headgroup chemistry (i.e., zwitterionic vs. anionic headgroups). On the basis of the observed differences, strategies are suggested for the most effective fluorotryptophan incorporation and application for 19F NMR studies of membrane protein topology, structural change, dynamics, and ligand binding. 2459-Pos Board B66 In Situ Solid-State Nmr Spectroscopy of APP Transmembrane Domain (TM) Structure and Dimerization in Native E. Coli Membranes Xiaoyan Ding1, Riqiang Fu2, Fang Tian1. 1 Penn State College of Medicine, Hershey, PA, USA, 2National High Magnetic Field Laboratory, Tallahassee, FL, USA. Cleavage of the amyloid precursor protein (APP) by a-, b- and g-secretases leads to generation of amyloid peptide (Ab), which is the primary constituent
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of Alzheimer’s disease (AD) plaques in the brain. Three consecutive GXXXG motifs and one GXXXA motif on the transmembrane domain (TM) of APP have been reported to be one or two important sites for mediating APP homodimerization in different experimental conditions, the difference are likely due to the membrane mimetic. We and several other groups recently demonstrated the feasibility to directly characterize recombinant proteins in native Escherichia coli (E. coli) membranes using magic-angle spinning (MAS) solid-state nuclear magnetic resonance (ssNMR)(2, 3). Here, we will present several strategies to further improve spectral sensitivity and resolution, and suppress background signals of E. coli proteins and lipids, including preparation of E. coli inner membranes, reverse 13C- and 15N-isotope labeling, and selective signal filtering with the frequency-selective REDOR pulse sequence. These improvements have allowed us to characterize the structure and dimerization of APP TM and its variants in a native membrane environment. 2460-Pos Board B67 Structural and Dynamical Basis of Protein Kinase C Alpha Regulation by the C-Terminal Tail Yuan Yang1, Julia A. Callender2, Alexandra C. Newton2, Tatyana I. Igumenova1. 1 Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA, 2Department of Pharmacology, University of California at San Diego, La Jolla, CA, USA. Protein Kinase C (PKC) isoenzymes are dynamic multi-modular proteins that regulate signal transduction processes at the membrane surface. When activated by second messengers, such as Ca2þ and diacylglycerol, PKC undergoes a drastic conformational transition from the inactive cytosolic state to the activated membrane-bound state. The structure of either state of PKC is not known. Using NMR, we demonstrate that the isolated Ca2þ-sensing lipid-binding C2 domain of PKCa interacts with the C-terminal tail of the kinase. We determined the structure of the complex between the C2 domain and the peptide corresponding to the phosphorylated hydrophobic motif of the C-terminal tail. The structure shows that the hydrophobic motif binds to the conserved lysine-rich cluster region of C2. The interface is stabilized by electrostatic interactions and stacking of the aromatic sidechains. We validated our structural model by mutating the interacting residues in full-length PKC, and characterizing these variants using in vitro FRET experiments and FRET-detected membrane translocation experiments in live cells. In addition, NMR-detected binding studies revealed that the hydrophobic motif and Ca2þ synergistically enhance each other’s affinities to C2. We propose a model where the C-terminal tail plays a dual role in PKC regulation: auto-inhibitory, through its interaction with the lysine-rich cluster of the C2 domain; and activating, through sensitization of PKC to intracellular Ca2þ oscillations. Support: Welch Foundation grant A-1784 (TII), NSF CAREER award CHE-1151435 (TII), NIH GM108998 (TII), and NIH GM43154 (ACN). 2461-Pos Board B68 Site-Directed Spin Labeling EPR Spectroscopy of the Cytoplasmic Tail of Influenza a M2 Alice L. Herneisen, Grace Kim, Kathleen P. Howard. Swarthmore College, Swarthmore, PA, USA. The M2 protein is a 97 residue homotetrameric, multifunctional ion channel that plays critical roles during the influenza infection cycle. While a variety of high-resolution biophysical techniques have been used to characterize the transmembrane domain (residues 22-46) and the juxtamembrane C-terminal region (46-62), less is known about the conformation and dynamics of the remaining residues of the C-terminal cytoplasmic tail. Here, we use site-directed spin labeling electron paramagnetic spectroscopy (SDSL-EPR) experiments to probe the secondary structure and membrane topology of cytoplasmic tail residues 60-80 when the protein is reconstituted into lipid bilayers. Cholesterol is essential for the role the C-terminal domain of the M2 protein plays in viral budding. SDSL-EPR data is collected in both in the presence and absence of cholesterol. 2462-Pos Board B69 Polarity and Charge as Determinants for Translocase Requirement for Membrane Protein Insertion Balasubramani Hariharan1, Raunak Soman2, Ross E. Dalbey3. 1 Biophysics Graduate program, The Ohio State University, Columbus, OH, USA, 2OSBP, The Ohio State University, Columbus, OH, USA, 3The Ohio State University, Columbus, OH, USA. Membrane protein biogenesis in bacteria is a well-studied but poorly understood area in the field of membrane proteins. In bacteria, the Sec translocon and the YidC insertase promote the membrane insertion process. Both proteins are essential, and universally conserved, and together insert about 95% of the proteins in E.coli. Membrane insertion of SecYEG-
dependent proteins likely occurs at the lateral gate of SecY, the channelforming subunit of the bacterial Sec translocon. On the other hand, YidC catalyzes insertion by possessing a hydrophilic groove, which can accommodate the hydrophilic region of the substrate that inserts across the membrane. Recently, Soman et al (2014) have reported that increasing the charge of the translocated region of procoat can route substrates from the YidC pathway into the Sec pathway. Here, we have further tested this polarity/charge hypothesis by showing that the major coat protein of bacteriophage M13 can become increasingly YidC/Sec dependent by making the periplasmic loop highly polar in the absence of charged residues. We also show that adding hydrophobic amino acids to highly polar or charged loop can decrease the Secdependence of the otherwise strictly Sec-dependent substrates. Additionally we demonstrate that increasing the driving force of insertion by adding four leucyl residues to the transmembrane segment leads to translocation of a highly charged region that was not inserted by native TM segment. Finally, we show that the length of procoat loop is a determinant for Sec dependent insertion. The combined results support the polarity/charge hypothesis and is consistent with the notion that membrane insertion occurs at the interface of SecYEG and YidC. 2463-Pos Board B70 Bip Binding Affects Integration of Transmembrane Domains Mirjam Zimmermann1,2, Marco Janoschke2, Martin Spiess2. 1 Molecular and Membrane Biophysics, Institute of Biophysics, Linz, Austria, 2 Biozentrum, Basel, Switzerland. The Sec61 translocon mediates translocation of proteins into the endoplasmic reticulum (ER) and enables integration of hydrophobic (H) segments into the lipid bilayer via its lateral gate. The luminal chaperone BiP (Kar2 in yeast) has been shown to act as a molecular ratchet, recruited to the emerging polypeptide by the J-domain of Sec63, and to be involved in post- and cotranslational translocation. In this study, we address the question whether BiP binding to the nascent chain affects the process of membrane integration vs. translocation, i.e. whether integration is governed by thermodynamic equilibrium between pore and membrane. To determine the hydrophobicity threshold of membrane integration, a series of model proteins based on the sequence of dipeptidylaminopeptidase B (DPAPB) with oligo-alanine H-segments containing increasing numbers of leucines. The translocated loop preceding the H-segments was replaced by a sequence of similar length containing multiple copies of segments known to bind BiP with high affinity or not to interact with BiP. While H-segments preceded by the DPAPB sequence required ~3.7 Leu for 50% integration, this threshold was reduced to ~2.1 Leu with the BiP-binding sequence and increased to ~5.2 Leu with the non-binding sequence. These results show that membrane integration of an H-segment is influenced by the already translocated sequence preceding it and suggest that BiP binding promotes membrane integration. We observed changes of the apparent free energy in integration between 1 to 3 kcal/mol. We propose a model in which Brownian motion of the translocated loop with bound chaperones preferentially promotes release of the H-segment into the lipid bilayer. The work was supported by grant W0125 of the Austrian science fund (FWF). 2464-Pos Board B71 Elucidating the Uncoupling of ATP Hydrolysis and Ca2D Transport in SERCA by Sarcolipin Erin Birdsall1, Alysha Dicke1, Gianluigi Veglia1,2. 1 BMBB, University of Minnesota, Minneapolis, MN, USA, 2Chemistry, University of Minnesota, Minneapolis, MN, USA. The activity of the sarco(endo)plasmic reticulum Ca2þ -ATPase (SERCA), which plays a key role in muscle contraction and relaxation, is regulated by sarcolipin (SLN), a 31-residue transmembrane peptide. Sarcolipin has been shown to uncouple ATP hydrolysis and Ca2þ transport in SERCA and is largely found in skeletal muscle and the atria of the heart. The interaction between SLN and SERCA is thought to play an important role in energy metabolism. SLN is composed of a transmembrane helix flanked by two short, unstructured Nand C-termini. The N-terminus is hypothesized to play a role in the uncoupling of SERCA, while the role of the C-terminus has been shown to be largely responsible for SERCA inhibition. In this study, we made two constructs of SLN, with either the C- or N-terminus deleted. We then used coupledenzyme activity assays to measure the effect of the SLN constructs on SERCA ATPase activity and isothermal titration calorimetry (ITC) to monitor the change in heat release due to ATP hydrolysis. We found through these methods that the deletion of the termini led effected the uncoupling function in SLN. The results of this study, in combination with forthcoming solid-state NMR studies, will help to establish the mechanism by which SLN regulates SERCA activity.