(581e) Investigating Orientation and Conformation of α-Helix and β-Sheet Model Peptides with Surface Analysis Techniques

Interactions between proteins and surfaces are critical to the success or failure of implants in the body. When adsorbed onto a synthetic surface, proteins often denature which can trigger the foreign-body response. It is therefore essential to develop methods to examine these interfacial phenomena. As the structure of proteins at interfaces is poorly understood, we have chosen to begin this study with peptides that form α-helix and β-sheet structures. These peptides will be absorbed to and characterized on self-assembled alkylthiol monolayers on gold surfaces. These SAMs form well-defined structures with surface properties can be systematically varied by changing the ω-functional group. The α-helix peptide is a 14-mer made up of lysine (K) and leucine (L) residues with a hydrophobic periodicity of 3.5. The β-sheet peptide is a 15-mer also made up of lysine and leucine with a hydrophobic periodicity of 2. Both peptides are made such that the hydrophobic side-chains are on one side of the peptide and the hydrophilic side chains are on the other. The SAMs had ω-functional groups of ?CH₃ and -COOH. The protein-surface interactions were first characterized by measuring adsorption isotherms on the various surfaces using X-ray photoelectron spectroscopy (XPS) and surface-plasmon resonance spectroscopy (SPR). The amount of peptide adsorbed was tracked by the nitrogen atomic percent for XPS and by the change in refractive index for SPR. These isotherms show that adsorption depends on the surface chemistry. The α-helix peptide forms a monolayer on the carboxylic-acid terminated SAM when adsorbed from a solution concentration that is 50 times lower than required to form a monolayer on the methyl-terminated surface. The SPR results showed that a portion of peptides were adsorbed reversibly and had a quantifiable disassociation rate. To see if the lysine or leucine residues were interacting with the surface, secondary ion mass spectrometry (SIMS) and sum-frequency generation (SFG) experiments were performed. SIMS experiments detected a difference in the ratio of the lysine characteristic mass peak (84) and the leucine characteristic mass peak (86) on the two surfaces. Principal component analysis (PCA) of the SIMS data confirmed that different side-chains were pointing away from the two SAM surface. SFG showed ordering of the methyl stretches of the leucine side-chains on deuterated ?CD₃ SAMs, and ordering of the amine stretches on the ?COOH SAMs. The phase of the SFG signal confirmed the orientation of the side-chains on the surface. Near-edge X-ray absorption spectroscopy (NEXAFS) was used to verify the orientation of these proteins on the surface. Analysis of the N K-edge of the adsorbed proteins on SAMs showed that the peptides were lying down on the surface. However, the degree of order for the α-helix was less than for the β-sheet.