SFG analysis of proteins at interfaces
Dr. Tobias Weidner
Understanding proteins on surfaces plays a key role for rational implant design, drug delivery, biomaterials, biomineralization and protein arrays development. Sum frequency generation (SFG) vibrational spectroscopy, owing to its unique interface sensitivity, can probe protein monolayers in situ under biologically relevant conditions. SFG is becoming very popular in the field because it can address a wide range of important questions: What is the protein conformation on the surface surface? Is the structure changing upon surface binding? How are proteins assembling into ordered films? SFG can also provide insight into orientation surface binding.
Fig. 1: Schematic drawing of SFG sample setup and polarization dependence of the protein backbone signal.
Figure 1 shows a typical sample setup for SFG protein studies. Films on surfaces or incorporated into artificial membranes can be deposited onto the prism and then probed in situ in a near-total internal reflection geometry. The secondary structure and orientation of proteins on surfaces can be probed using amide I SFG vibrational spectra collected with different polarization combinations of the incident fundamental beams and the detected SFG photons. The graph in Fig 1 c) plots the calculated amide I intensity ratio of two polarization combinations for different α-helix orientations with respect to the surface.
If combined with isotopic labeling, SFG can probe the orientations of individual side chains within adsorbed proteins. When hydrogen is replaced by deuterium in a C-H bond, the respective stretching modes are red-shifted by about 800 cm-1 and the labeled side chain can be analyzed without spectral confusion with background C-H resonances. This approach is particularly powerful when combined with complementary techniques such as solid-state NMR, near-edge X-ray absorption fine structure (NEXAFS) spectroscopy and neutron reflectometry. Figure 2 shows the structure of a model peptide adsorbed onto polystyrene. The orientations of all binding side chains have been determined by combining SFG and solid-state NMR measurements. We will now extend this approach to more complex, 'real-life' proteins such as biomineralization peptides, covalently immobilized functional proteins and membrane proteins.
Fig. 2: Lysine-leucine model peptide on polystyrene. Leucine interacts with the hydrophobic surface. The secondary structure and orientation can be determined using SFG and NEXAFS. Right: SFG spectrum of Leu 12 (labeled red) after site directed deuterium substitution used for structural analysis. The orientations of all leucine side chains were determine using a combination of SFG and solid-state NMR.
T. Weidner, N. F. Breen, K. Li, G. P. Drobny, D. G. Castner. Sum Frequency Generation and Solid-State Nuclear Magnetic Resonance Study of the Structure, Orientation and Dynamics of Peptides Adsorbed onto Polystyrene Surfaces, Proceedings of the National Academy of Sciences of the USA, 107, 13288 (2010).
T. Weidner, J. S. Apte, L. J. Gamble, D. G. Castner. Probing Orientation and Conformation of α-Helix and β-Strand Model Peptides on Self-Assembled Monolayers Using Sum Frequency Generation and NEXAFS Spectroscopies, Langmuir 26, 3433 (2010).
L. Baugh, T. Weidner, P.-C. T. Nguyen, J. E. Baio, L. J. Gamble, D. G. Castner, P. S. Stayton. Probing the Orientation of Surface-Immobilized Protein G B1 using ToF-SIMS, Sum Frequency Generation, and NEXAFS Spectroscopy, Langmuir, 26, 16435 (2010).
T. Weidner, N. T. Samuel, K. McCrea, L. J. Gamble, R. S. Ward, D. G. Castner. Assembly and Structure of α-Helical Peptide Films on Hydrophobic Fluorocarbon Surfaces, Biointerphases 5, 9 (2010).
N. F. Breen, T. Weidner, K. Li, D. G. Castner, G. P. Drobny. A Solid-State Deuterium NMR and SFG Study of the Side Chain Dynamics of Peptides Adsorbed onto Surfaces, Journal of the American Chemical Society 131, 14148 (2009).