ABSTRACTS Applications of Solid-State Nuclear Magnetic Resonance (SSNMR) and Neutron Reflectivity (NR) to the Investigation of Protein/Surface Interfaces

Applications of Solid-State Nuclear Magnetic Resonance (SSNMR) and Neutron Reflectivity (NR) to the Investigation of Protein/Surface Interfaces Wendy Shaw, Kim F. Ferris, Susan Krueger, Ursula Perez-Salas, Vitalii Silin, Duncan J. McGillivray, Allison A. Campbell, Michael L. Paine, and Malcolm L. Snead Pacific Northwest National Laboratory, Richland, WA; National Institute of Standards and Technology, U.S. Department of Commerce, Gaithersburg, MD; University of California, Irvine, Irvine, CA; Department of Biophysics, Johns Hopkins University, Baltimore, MD; University of Southern California, Los Angeles, CA Introduction Naturally occurring biominerals possess an impressive array of strength, order, and nanostructure, but the protein-mineral interactions that result in these properties are not well understood; as such, this is a primary goal. The secondary structure and orientation of biomineralization proteins are thought to play an important role in the formation of biominerals; however, there is little experimental evidence to explain the underlying mechanisms. SSNMR and NR have the potential to elucidate site-specific structure and orientation information of biomineralization proteins, inaccessible by other methods, and are highlighted in an investigation of the protein-crystal interaction of an enamel-forming protein. Methods to investigate crystal nucleation and growth also are presented. Purpose This research aims to probe the protein orientation and structure of an amelogenin protein, leucine-rich amelogenin peptide (LRAP) on the hydroxyapatite (HAP) surface. Methods SSNMR, NR, and computational methods were utilized to study the surface-immobilized LRAP protein. The structure of the N-terminus was studied, and the protein-surface interaction of the C-terminus was investigated. Results These experiments demonstrate that the C-terminus is oriented next to the HAP surface. NR measurements yielded a distance from the deuterated region to the COO terminated surface of 5-10Å, in good agreement with the SSNMR data, which yielded a Cala46-P distance of 7-7.5Å from the labeled alanine to the hydroxyapatite surface. Modeling results are consistent with experimental measurements, resulting in an average Cala46-P distance of 7.3Å for LRAP oriented onto the 100 face of the HAP. Structural measurements using SSNMR indicate a significant change in the structure of the N-terminus upon binding to the HAP surface. Conclusions These results demonstrate the unique capability of SSNMR, NR, and computational modeling to provide quantitative, site-specific, molecular-level detail of the protein-surface interface.