Code: TOE	Time Slot/Poster Number: 4:00-4:25	Session: Solids Bio-Applications
Magic-Angle Spinning Approaches to Structures of Membrane Proteins and Fibrils
Chad Rienstra
U. of Illinois, Urbana, IL
Abstract
We present methodology and results aimed at the goal of de novo structure determination of large, non-crystalline solid proteins. Here we focus discussion on the progress towards complete resonance assignments and collection of structural restraints for alpha-synuclein fibrils (14 kDa) and the DsbA-DsbB membrane protein complex (41 kDa). Methods for enhancing sensitivity include proton detection and new probe technologies, enabling studies near the physiological temperatures at which spectral resolution is often optimal.

Code: TOE	Time Slot/Poster Number: 4:25-4:40	Session: Solids Bio-Applications
High-resolution structure of a drug-complexed proton channel in lipid bilayers from solid-state NMR
Sarah D. Cady1; Klaus Schmidt-Rohr1; Wenbin Luo1; Jun Wang2; Cinque S. Soto2; William F. DeGrado2; Mei Hong1
1Iowa State University, Ames, IA; 2University of Pennsylvania, Philadelphia, PA
Abstract
The high-resolution structure of the drug-complexed influenza M2 proton channel is determined using solid-state NMR. Using multi-spin 13C-2H REDOR, we show that two amantadine-binding sites exist in M2 in phospholipid bilayers. The high-affinity site, occupied by a single amantadine (Amt), is located in the N-terminal channel pore. The 15 deuterons of perdeuterated Amt speeded up REDOR dephasing of the 13C-labeled peptide, allowing distance quantification that resulted in a 0.3 Å-resolution solid-state NMR structure of the drug-complexed M2 at high pH in lipid bilayers. Under excess Amt concentrations, a second, lower-affinity, binding site was also observed. 2H NMR lineshapes reveal different orientation and dynamics of the drug in the two binding sites. Water-protein 1H spin diffusion experiments show that Amt reduces the water accessibility of pore-facing residues in the middle of the transmembrane helix, consistent with the pore location of the high-affinity binding site. 3D lattice simulations of the spin diffusion buildup indicate that the water-exposed protein surface area is reduced by 50% from the low-pH open state to the high-pH drug-bound state. These results indicate that amantadine inhibits the influenza M2 proton channel by physical occlusion and interruption of the water wire. The study demonstrates the ability of solid-state NMR to elucidate small-molecule interactions with membrane proteins and determine high-resolution structures of their complexes directly in lipid bilayers.

Code: TOE	Time Slot/Poster Number: 4:40-5:05	Session: Solids Bio-Applications
Cobalt MMP: Pseudocontact Shifts to determine Atomic Coordinates in the Solid State
Claudio Luchinat
CERM, Florence, Italy
Abstract
Pseudocontact shifts (PCS) in paramagnetic molecules originate from the magnetic susceptibility tensor anisotropy of the paramagnetic center (usually a metal ion). PCS have been used since 1996 as structural restraints in metalloprotein structure determination in solution. Their contribution to the improvement of the structure quality is significant, even in the presence of many NOEs, because of their long-range nature. It has now been shown that PCS can be accurately determined also in the solid state, and their relative importance is even higher because NOE-like restraints are fewer. PCS also provide information on atomic coordinates of neighboring molecules in the crystal. Finally, advancements in theory and hardware allow us to observe nuclei closer and closer to the metal ion.

Code: TOE	Time Slot/Poster Number: 5:05-5:20	Session: Solids Bio-Applications
Purely Spectroscopic Assignment of Solid-State NMR Spectra of Magnetically Aligned Pf1 Phage Using Mismatched Hartmann-Hahn Magnetization Transfer
Robert W. Knox; Alexander A. Nevzorov
North Carolina State University, Raleigh, NC
Abstract
A purely spectroscopic means of assignment remains an outstanding problem in NMR of oriented membrane proteins. Here we implement the recent method based on the transfer of magnetization between the low spins via the proton bath under mismatched Hartmann-Hahn conditions combined with a separated-local field experiment. The arising cross-peaks establish connectivity between the 15N spins of the backbone, thus providing a new method for assignment. Uniformly 15N-labeled Pf1 phage (provided by S. J. Opella, UCSD) has been used to test the applicability of the method. About 80% of the original assignments have been confirmed, which results in only 1.3 Angstrom backbone rmsd relative to the original structure (1ZN5). Moreover, the present method does not require multiple selectively labeled samples.

Code: TOE	Time Slot/Poster Number: 5:20-5:45	Session: Solids Bio-Applications
Solid State NMR Studies of Biomaterials: The Structure of a Surfaced-Adsorbed Protein
Gary Drobny
University of Washington, Seattle, WA
Abstract
The long-term objective of our research is to elucidate the molecular recognition mechanisms used by proteins to control biomineralization processes. A variety of interesting proteins that are found in mineralized tissues act as nature's crystal engineers, where they control the growth of inorganic composites such as hydroxyapatite (HAP) (the mineral phase found in bone/teeth). A particularly important class of acidic proteins found in hard tissues is known to regulate normal hard tissue formation and remodeling, and they are also involved in pathological processes such as dental caries, kidney stone formation and arterial calcification. However, due to the difficulties in studying the protein structure and function at inorganic solid surfaces, there is still remarkably little known of the molecular structure-function relationships governing hard tissue engineering. Our group has been developing and applying solid-state NMR (ssNMR) techniques together with advanced computational methods to determine protein structure and dynamics on their biologically relevant hydroxyapatite surface, together with the inter-related mechanistic characterization of hydroxyapatite recognition and crystal growth dynamics. In this talk we will present a full three-dimensional statherin structural model based on NMR experimental constraints, that connects the molecular mechanisms underlying hydroxyapatite adsorption thermodynamics and crystal engineering function. This molecular insight is being used in outside but related projects in our group to design biomimetic peptide coatings for biomaterial/tissue engineering applications, and could provide new routes to inhibiting the bacterial adhesion to material surfaces.