AVS 56th International Symposium & Exhibition
    Biomaterial Interfaces Tuesday Sessions
       Session BI-TuM

Paper BI-TuM9
Probing Individual Side-Chains of Peptides at Interfaces Using Isotope Labeling with Sum Frequency Generation Spectroscopy

Tuesday, November 10, 2009, 10:40 am, Room K

Session: Proteins and Cell Interactions at Interfaces II
Presenter: T. Weidner, University of Washington
Authors: T. Weidner, University of Washington
N.F. Breen, University of Washington
G.P. Drobny, University of Washington
D.G. Castner, University of Washington
Correspondent: Click to Email
Controlled immobilization of peptides onto artificial biointerfaces plays a key role in antifouling, implant and immunosensor technologies and it is of crucial importance to develop tools to examine interfacial properties of adsorbed peptides. Sum frequency generation (SFG) spectroscopy can probe biomolecules at the solid-liquid interface. Isotope labeling can address specific protein regions but its potential in conjunction with SFG spectroscopy has remained mostly unexplored. We combine these techniques to characterize the structure of synthetic model peptides on surfaces in PBS buffer. The peptide used is a 14-mer of hydrophilic lysine (K) and hydrophobic leucine (L) residues with an α-helical secondary structure. These LK14 peptides have the hydrophobic side-chains on one side of the helix and the hydrophilic on the other. Deuteration of the isopropyl group of each of the leucines, totaling 8 samples, was used to probe individual leucine side chains of LK14 adsorbed onto a hydrophobic polystyrene surface. Side-chain orientations were determined using ratios of the asymmetric CD3 stretching mode at 2221 cm-1 acquired with different polarization combinations. We found that the orientation of the leucine side chains in the surface-bound LK14 was remarkably different from the calculated solution structure. Leucines in the center of the peptide are more oriented towards the surface while those at the ends of the amino-acid sequence are more bent away, indicating the leucines in the center of the peptide chain play a dominant role for the binding of the peptide. Solid state NMR data acquired on polystyrene beads is in line with the determined orientation change upon binding. In addition, 15N labeling was used to address the controversial assignment of a pronounced peak near 3300 cm-1 observed for a variety of proteins adsorbed onto surfaces. This spectral feature has been assigned to both N-H containing side chains and backbone-related amide A resonances before. 15N labeling of the lysine side chains resulted in a 9 cm-1 red-shift of this peak in the spectrum of LK adsorbed onto a SiO2 surface, showing the 3300 cm-1 feature is related to the terminal amine group on the lysine side chains.