is temperature and almost quantitative refolding of the KvAP channel is ob- served at 80 o C. In order to differentiate between insertion into the bilayer and folding within the bilayer, we developed a cysteine protection assay. Using this assay, we demonstrate that insertion of the unfolded protein into the bilayer is relatively fast at room temperature and independent of lipid composition sug- gesting that temperature and bilayer composition influence folding within the bilayer. Further, we demonstrate that in vitro folding provides an effective method for obtaining high yields of the native channel. Our studies suggest that the KvAP channel provides a good model system to investigate the folding of a multi-domain integral membrane protein. 1345-Pos Board B115 A Homology Modeling-Simulation Protocol for Construction and Assess- ment of Hv1 Models Kethika Kulleperuma 1,2 , Susan M.E. Smith 3 , John Holyoake 2 , Nilmadhab Chakrabarti 2 , Deri Morgan 4 , Boris Musset 4 , Thomas E. DeCoursey 4 , Vladimir V. Cherny 4 , Re ´gis Pome `s 1,2 . 1 University of Toronto, Toronto, ON, Canada, 2 Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, Canada, 3 Department of Pathology, Emory School of Medicine, Atlanta, GA, USA, 4 Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL, USA. The voltage-gated proton channel (Hv1) of leukocytes, basophils, airway epithe- lium, and spermatozoa is extremely selective. Hv1 presents structural and func- tional similarities to the voltage-sensor domain (VSD) of voltage-gated potassium (Kv) and sodium (Nav) channels. Hv1 and the VSDs of Kv and Nav sense changes in membrane potentials and contain four a-helical segments as well as conserved arginine residues on the fourth helix. Although the electrophys- iological features of Hv1 are well characterized, the molecular mechanism of pro- ton conduction is unknown. This is largely because an experimentally-determined structure of Hv1 is not available. An alternative source of structural information is homology modeling, whereby a model of Hv1 can be constructed using the atomic structures of the VSDs of Kv and Nav as templates. However, since the sequence identity between Hv1 and templates is below 20%, a critical assessment of gen- erated models is essential. Here we present a homology modeling-simulation strategy using alternative sequence alignments to construct, assess, and validate alternative homology models. Comparison of a range of structural properties be- tween the models and templates is used to guide the selection of an optimal model for Hv1. This method represents a generalized strategy that can be applied to other membrane proteins that lack high sequence identity to their templates. 1346-Pos Board B116 Contributions of the Inner Cavity to the Resistance of High Conductance KD Channels Determined with a Two-Resistor Model Yanyan Geng, Karl L. Magleby. Miller School of Medicine University of Miami, Miami, FL, USA. Both BK and MthK are high conductance Kþ channels. The dimensions of the conduction pathway for MthK are known from crystal structures. BK’s conduc- tion pathway may have similar dimensions. Geng et al. (2011) changed the size of the entrance to the inner cavity of BK channels by substituting uncharged amino acids with different sized side chains at positions E321/E324 located at the entrance. Fitting plots of conductance vs. amino acid side chain volume with a two-resistor model, where R1þR2 is the total resistance of the conduc- tion pathway, suggested that ~7% of the total resistance for wt channels was at the entrance to the inner cavity (R2), with ~93% arising from the remainder of the conduction pathway including the selectivity filter (R1). Shi et al. (2011) changed the size of the conductance pathway deep in the inner cavity of MthK by substituting amino acids at A88. Our analysis of their data with the two-resistor model suggests that the deep segment of the inner cavity encircled by A88 (R2) contributes >80-90% of the total resistance of the condution path- way. On this basis, the deep inner cavity leading to the selectivity filter would constitute the major source of resistance. Alternatively, because the deep inner cavity has a narrow diameter compared to the entrance to the inner cavity, the substitution of larger amino acids at A88 in MthK may constrict the diameter sufficiently so that Kþ has to start shedding waters of hydration before it rea- ches the selectivity filter, leading to an artificial high resistance localized deep in the inner cavity that would not be present in wt channels. NIH AR32805. 1347-Pos Board B117 Spectroscopic Insights into the Structural Dynamics and Mechanism of Ionophore Function of Valinomycin in Lipid Membranes Christopher M. Halsey, Derek A. Benham, Renee D. JiJi, Jason W. Cooley. University of Missouri, Columbia, MO, USA. The ionophore valinomycin has been well studied as a model carrier depsipep- tide, whose proposed mechanism involves the selective complexation of a mono- valent cation in solution which is carried across the membrane and released. However, the proposed mechanisms of transport rely primarily on studies done in non-polar organic solvent. Here we present the first spectroscopic studies of valinomycin aimed at structural information done in the presence of a model lipid membrane without the need for sample or solvent modification. Deep-UV reso- nance Raman spectroscopy with a 197-206 nm excitation range is ideally suited to probe the secondary structure without inter- ference from the lipid moiety. Two lipid en- vironments were investigated, micelles and single unilamellar vesicles, which induce different conformations of valinomycin. Complexation of valinomycin with potas- sium was also investigated, both with and without competing sodium ions present. Im- plications for the mechanism of ion trans- port are discussed. 1348-Pos Board B118 Complete Dissociation of an ATP-Binding Cassette Nucleotide-Binding Domain during the Hydrolysis Cycle Maria E. Zoghbi, Srinivasan Krishnan, Guillermo A. Altenberg. Texas Tech University Health Sciences Center, Lubbock, TX, USA. ATP-binding cassette (ABC) proteins catalyze the transport of substrates across membranes. Many ABC proteins, including P-glycoprotein, pump substrates out of the cells. There are two competing models to explain the mechanism of ABC exporters: 1) AThe alternating-access model, where large conforma- tional changes take place during the transport cycle as a consequence of ATP-induced nucleotide-binding domain (NBD) dimerization. 2) An alterna- tive model, which suggests that the power stroke triggered by ATP binding con- sists of only moderate rearrangements, with the NBDs in contact at all times during the transport cycle. Here, we performed experiments on isolated bacte- rial NBDs using luminescence resonance energy transfer (LRET) and trypto- phan fluorescence spectroscopy, to assess monomer association/dissociation in real time. We used MJ0976, a NBD from the thermophile M. jannaschii, as a model. We found that: 1) The dependence of dimerization for MgATP is stronger (Kd of ~ 5 mM) than that for NaATP (~80 mM). 2) The rate of di- merization increased >10-fold in MgATP vs. NaATP. 3) A steady-state dy- namic equilibrium is reached in MgATP, with ~50% of the protein in dimeric form. 4) The dimers completely dissociate into monomers during the hydrolysis cycle. Our observations support ABC protein models involving NBDs association/dissociation. This work was supported grants from CPRIT (RP101073) and NIH (R01GM79629 and R01GM079629-03S1R01). 1349-Pos Board B119 Mapping the Backbone Dynamics of the Alzheimer’s APP Transmem- brane Domain Oxana Pester 1 , Daniel Hornburg 1,2 , Milena Duerrbaum 1 , Philipp Hornburg 1 , Christina Scharnagl 1 , Gerd Multhaup 3 , Dieter Langosch 1 . 1 Technische Universita ¨t Mu ¨nchen, Freising, Germany, 2 Department of proteomics and signal transduction, MPI f. Biochemie, Martinsried, Germany, 3 Freie Universita ¨t Berlin, Berlin, Germany. The Amyloid Precursor Protein (APP) plays a central role in Alzheimer’s dis- ease. Its dimeric transmembrane domain (APP-TMD) is subject to several con- secutive cleavage events by the gamma-secretase leading to a certain product pattern of amyloidogenic peptides (Munter et al., 2007). Here, we address the question how backbone dynamics of the alpha-helical APP-TMD is mapped. We have investigated the backbone dynamics of several APP-TMD model pep- tides with hydrogen-deuterium exchange (HDX) experiments coupled to mass spectroscopy. The underlying principle is the exchange of amide-protons for deuterons which is dependent on the dynamics of the alpha-helix. The investigation demonstrates that the N-terminal part of the APP transmem- brane domain exhibits higher backbone dynamics than the C-terminal part. Fur- ther results show that exchanging threonines for valines along the APP-TMD sequence changes the helix backbone flexibility. To characterize the dynamics of each cleavage region along the APP-TMD a hybrid peptide set was exam- ined. The hybrid peptides consist of a constant poly-leucine sequence and a varying 8 residue long APP-TMD-region where the last and last but two/ three residues represent cleavage sites. To this end, we could discover local differences in APP-TMD helix dynamics. The findings are discussed in terms of substrate recognition and binding and could explain the A beta product pattern in gamma-secretase proteolysis. Munter, L. M., Voigt, P., et al. (2007). ‘‘GxxxG motifs within the amyloid pre- cursor protein transmembrane sequence are critical for the etiology of A beta 42.’’ EMBO Journal 26(6): 1702-1712. 266a Monday, February 27, 2012