Dynamic Conformational Changes in a Cyclic Nucleotide-Binding Domain

Monday, February 13, 2017 Nitric oxide synthase (NOS) generates a signaling molecule, nitric oxide, which is essential for processes such as vasodilation and neurotransmission. The enzyme forms homodimers containing cofactors FAD, FMN, and heme, each housed in subunits of the enzyme. Synthase activity in NOS is activated by calmodulin (CaM), which binds to a region adjacent to the FMN domain. Conformational motion is necessary to position sequential electron donoracceptor pairs adjacent to one another for transfer of electrons from the FAD domain to the FMN domain and then to the oxygenase domain of the opposite member of the homodimer. We characterized the conformations and dynamics of endothelial and neuronal NOS with bound CaM by time-resolved and singlemolecule detection of fluorescence from a fluorophore attached to CaM. Maximum entropy analysis of fluorescence decays for both neuronal and endothelial NOS reveals four conformational states, resolved by their distinct extents of quenching by energy transfer to heme. Clues to the identity of each population are found from their dependence on calcium concentration and from mutations known to disrupt specific interactions. The CaM mutation K115E, known to disrupt docking of CaM to the oxygenase domain, abolishes the two shortest lifetime populations. Single-molecule trajectories reveal conformational interchange on the millisecond to second time scales and permit analysis of the rates of interchange of conformational states. Exchange appears slowest to and from the conformation with highest quenching, consistent with the identification of this conformation as the ‘‘output’’ state of the enzyme. 1002-Pos Board B70 Probing the Conformational Dynamics of Butyrophilin 3A1 using Atomic Force Microscopy and Molecular Dynamics Simulations Christopher T. Boughter1, Benoit Roux2, Erin J. Adams2. 1 Biophysical Sciences, University of Chicago, Chicago, IL, USA, 2 Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA. In certain types of cancers and pathogenic infections such as Mycobacterium tuberculosis an accumulation of pyrophosphate metabolites, collectively called phosphoantigens (pAgs), in the unhealthy cell can lead to immune recognition and response. Vg9Vd2 T-Cells are primarily responsible for this recognition, and Butyrophilin 3A1 (BTN3A1) has been identified as a key mediator in this pAg induced response. Recent progress demonstrates that pAg binds to the intracellular domain of BTN3A1 resulting in stimulation of the T-Cell. The sequence of events that connects pAg binding to this stimulation remain unclear. Previous crystallographic and cell biological studies have provided starting hypotheses, focusing mainly on conformational change and membrane immobilization. To further probe this system and test these hypotheses, atomic force microscopy (AFM) and molecular dynamics (MD) simulations have been employed to elucidate the dynamics of the pAg binding pocket and the subsequent conformational changes associated with this binding. The primary focus of AFM experiments was to survey the topography of the extracellular domains on a supported bilayer with and without phosphoantigen. Molecular Dynamics simulations with explicit solvent focused on the intracellular ligand binding domain in an attempt to characterize both protein-protein and protein-ligand interactions. Together these physical techniques have started to shed light upon this complex biological system. 1003-Pos Board B71 Dynamic Conformational Changes in a Cyclic Nucleotide-Binding Domain Tobias Zbik. Center of Advanced European Studies and Research (Ceasar), Bonn, Germany. The understanding of protein function is still dominated by static pictures provided by NMR spectroscopy and X-ray crystallography. However, it is clear that the function of a protein is ultimately governed by its dynamic character. Addressing this aspect experimentally is challenging because available techniques are either limited to steady-state observations or rather suited for very particular systems. Here, we show how to study the dynamics of ligandinduced conformational changes in proteins. As a model system, we have chosen the cyclic nucleotide-binding domain (CNBD) from the CNG channel MloK1 from Mesorhizobium loti. Binding of cAMP to this CNBD leads to well characterized changes in the conformation of the protein as revealed by NMR and X-ray crystallography. To study the kinetics of these conformational rearrangements that ultimately lead from the open, ligand-free state to the closed, ligand-bound state of the protein, we are using DEER/PELDOR (Double Electron-Electron Resonance/Pulsed Electron Double Resonance). Combined with rapid mixing and freezing techniques, we are trying to derive a time-resolved picture of the structural transitions that occur upon ligand binding.

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Membrane Protein Folding 1004-Pos Board B72 The Significance of Surface Complementarity on the Free Energy of Membrane Protein Assembly in Membranes Kacey Mersch, Rahul Chadda, Venkatramanan Krishnamani, Marley Brimberry, Janice L. Robertson. University of Iowa, Iowa City, IA, USA. The dimerization interface of the 18-TM helix ClC-ec1 Cl-/Hþ antiporter is formed by four alpha helices (H, I, P and Q) that are lined entirely by nonpolar residues (leucines and isoleucines). The resultant surface fits well with its protein partner, presenting ClC-ec1 as an interesting model system to study the effect of shape complementarity on the stability of protein association. Recently, we established single-molecule fluorescence microscopy methods allowing for the measurement of the equilibrium dimerization reaction of ClC-ec1 in 2:1 POPE/POPG lipid bilayers. Here, we investigate the effect that cavities, engineered via subtractive substitutions to ALA, have on the free energy of dimerization in the lipid bilayer. We first engineered ClC constructs with large cavities by mutating entire helix surfaces to ALA, 4-5 residues at a time. Each of these constructs were stable and folded upon purification in detergent, and demonstrated comparable functional Cl- transport after reconstitution in membranes. However, while all-ALA helix I, P and Q are almost entirely dimeric as determined by size exclusion chromatography in detergent, all-ALA helix H shifts to a monomeric form. Further investigation of the residues on helix H indicates that L194A alone is responsible for the shift to the monomeric state. Cysteine accessibility and functional reconstitution experiments show that L194A is folded and functional in both monomeric and dimeric states. Measurement of the DDG of dimerization in 2:1 POPE/ POPG shows that L194A shifts stability by 2 kcal/mole, similar to the effect of a bulky I422W substitution at the interface. We conclude that L194 is an energetic hotspot on the dimerization interface, but the reason for this shift in stability remains unknown. Examination of the structure shows that L194 is positioned along the symmetry interface of the subunit fold suggesting that it may be involved in defining the shape of the dimerization interface, and thus mutation may alter shape complementarity by increasing dynamics of those helices. 1005-Pos Board B73 In vitro Reversible Folding of the Membrane Transporter LeuT in Membrane Mimetic Environments Michael R. Sanders. Chemistry, King’s College London, London, United Kingdom. Biological membranes provide a selective and chemically sealed barrier for cells. Transport of ions and small molecules across the membrane is mediated by transporter proteins and the breakdown of a cell’s ability to produce functionally folded membrane transport proteins can lead to dysfunction and has been implicated in many diseases. However, little is known about the processes that govern the folding of a-helical integral membrane proteins, taking into account that these proteins fold and maintain functional structures within membranes of various organelles. The neurotransmitter sodium symporter (NSS) protein family is an example of a-helical transporter proteins. The NSS family encompasses a wide range of prokaryotic and eukaryotic ion-coupled transporters that regulate the transport of neurotransmitter molecules whose dysfunction has been implicated in several neurological and psychiatric disorders. We have established 2 state folding mechanisms in detergent and reconstituted into lipid bilayers In vitro of LeuT; the prokaryotic orthologue of the NSS family from the organism Aquifex aeolicus. LeuT is responsible for the uphill transport of amino acids as a part of a symport mechanism driven by a sodium electrochemical gradient. In Vitro reversible folding of membrane transporters has been studied mostly within protein detergent complexes. We have demonstrated that two-state folding is also possible within a lipid bilayer environment without affecting the phase or structure of the bilayer. Biophysical techniques show that the stability of the protein in the phospholipid bilayer is dependent upon the phospholipid head group composition. Furthermore, the stability of LeuT reconstituted into the bilayer is heavily influenced by the addition of anionic charged head groups, which when coupled to increased lateral pressure results in a loss of two state folding. 1006-Pos Board B74 Monomeric Potassium Channels may Exist as Heterogeneous Ensemble before Assembly Kevin Song. Biophysical Sciences, University of Chicago, Chicago, IL, USA. For many potassium channels, tetramerization domains in either N- or C-terminal of the pore domain assist in tetramer assembly by increasing local