Keynote Room 1
Biosurfaces
Tue 14.30 – 15.10
15th European Conference on Applications of Surface and Interface Analysis 2013, ECASIA’13
Forte Village Resort, Sardinia, Italy, October 13 – 18, 2013
Strategies for Characterizing the Structure of Surface Bound Peptides and
Proteins
David G. Castner*
National ESCA and Surface Analysis Center for Biomedical Problems
Departments of Bioengineering and Chemical Engineering
University of Washington, Seattle, WA, 98195-1653 USA
*
Corresponding author: [email protected]
1. Introduction
Information about protein structure and function at
interfaces on the molecular level is crucial in drug
design, biosensor applications and biomaterial
engineering. Proteins on surfaces play an integral part in
many biomedical applications (implanted biomedical
devices, diagnostic arrays, tissue engineering scaffolds,
cell culture, etc.) as well as in biomimetic material
design strategies. This importance has stimulated
research towards developing techniques to assess the
structure, activity and surface interactions of
immobilized proteins. X-ray diffraction and solution
phase NMR methods are well established for
determining the structure of proteins in the crystalline or
solution phase. However, those techniques can’t be
used to characterize the structure of proteins at surfaces
and interfaces. To date the most successful method for
investigating the structure of surface bound proteins and
peptides has been to use a multi-technique approach.
The combination of protein engineering, experimental
methods (solid state NMR (ssNMR), sum frequency
generation
(SFG)
vibrational
spectroscopy,
time-of-flight secondary ion mass spectrometry
(ToF-SIMS), near edge x-ray absorption fine structure
(NEXAFS), etc.) and molecular dynamic (MD)
simulations has recently provided promising results.
Protein engineering is used to introduce selective
mutations and labels into the proteins and peptides.
The experimental methods then exploit those selective
mutations and labels to obtain detailed structure
information about the proteins and peptides (side chain
orientations, backbone orientations, secondary structure
determination, etc.). The experimental results are then
used to guide the MD simulations to obtain additional
atomic level information about the structure of surface
bound proteins and peptides.
2. Results
Results from a model α-helical and β–strand peptides
containing hydrophilic lysine (K) and hydrophobic
leucine (L) residues will be used to demonstrate surface
analysis strategies for obtaining detailed structural
information about immobilized biomolecules. For LK
peptides their backbones were oriented parallel to the
surfaces with the leucine side chains are oriented
towards hydrophobic surfaces such as methyl
self-assembled monolayers (SAMs) and polystyrene,
while the lysine side chains are oriented towards the
carboxylic acid terminated SAMs. A kinetic study of the
film formation process on a fluorocarbon surface
showed rapid adsorption of the peptides and ordering of
the leucine methyl groups followed by a longer
assembly process where the peptide backbone slowly
ordered. Selective, individual deuteration of the
isopropyl group in each leucine residue was used to
further probe the orientation and dynamics of each
individual leucine side chain of the LK α-helical peptide
adsorbed onto polystyrene. The orientation of each
leucine side-chain in the adsorbed peptides was
determined by SFG. These results were then correlated
with side chain dynamics determined by ssNMR
experiments.
NEXAFS spectroscopy with fluorine labeling was used
to quantify the orientation of the F7 and F14
phenylalanine rings in the 15 amino acid N-terminal
binding domain of statherin, SN15, adsorbed onto HAP
surfaces. The NEXAFS-derived phenylalanine tilt
angles have been verified with SFG vibrational
spectroscopy.
The
methodology
developed
for
structural
characterization of adsorbed peptide characterization
was then extended to a small rigid protein (Protein G B1
domain, 6kDa) bound via chemical and electrostatic
interactions to SAM surfaces. ToF-SIMS, which
sampled the amino acid composition of the exposed
surface of the protein film, used the relative enrichment
of secondary ions from amino acids located at opposite
ends of the proteins to describe protein orientation. SFG
spectral peaks characteristic of ordered α-helix and
β-sheet elements were observed for both systems and
the phase of the peaks indicated a predominantly upright
orientation for both the covalent and electrostatic
configurations. Polarization dependence of the
NEXAFS signal from the N 1s to π* transition of the
peptide bonds that make up the β-sheets also indicated
protein ordering at the surface.
This work is now being applied to characterize the
structure of Protein G immobilized onto SAM
functionalized gold nanoparticles.
3. Conclusions
A powerful set of surface analysis techniques
(ToF-SIMS, SFG, NEXAFS, ssNMR, XPS, etc.) have
been developed for characterizing the structure of
peptides and proteins bound to surfaces and interfaces.
The overall orientation of bound peptides and proteins
has been determined along with the interactions and
orientation of specific side chains with the surface.