Development Of Methods For Directed And Structured Immobilization Of Biomolecules

Author : Whitney April Fies
Publisher :
ISBN 13 :
Total Pages : 304 pages
Book Rating : 4.:/5 (114 download)

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Download or read book Creating and Characterizing Peptide Functionalized Surfaces for Protein Immobilization written by Whitney April Fies and published by . This book was released on 2019 with total page 304 pages. Available in PDF, EPUB and Kindle. Book excerpt: The specificity, efficiency, and broad-spectrum functionality of proteins make them desirable materials for use in a wide range of applications, including biosensors, biofuel cells, or drug delivery technologies. Capitalizing on proteins for these purposes often requires immobilizing proteins to inorganic surfaces, such as to an electrode for a biosensor or a substrate for heterogeneous catalysis. Successful immobilization of proteins requires the preservation of protein conformation and function in an environment completely different than the cell. Moreover, the stability of a biomolecule depends on the presence or absence of local water molecules in and around its structure. Despite this, conventional methods for characterizing biomolecules at inorganic surfaces do not give direct information about the amount of water at these interfaces and how that water affects biomolecule structure. Here, we have developed a strategy for protein immobilization intended to preserve native protein structure by creating a biomimetic surface that imitates a protein's natural cellular environment, and subsequently determined how water affects the structure of these surfaces. Biomimetic surfaces were made by covalently tethering short, helical peptides to an alkyl thiol self-assembled monolayer (SAM) on a gold substrate. Our helical peptides are designed to have solution-facing surfaces that attract and immobilize proteins of interest to the surface through an electrostatic mechanism. In order to investigate the importance of water in the structure and function of these biomimetic surfaces, we measured the quantity of water within the peptide and SAM layers using neutron reflectometry (NR) with the Liquids Reflectometer at Oak Ridge National Laboratory. Prior to peptide functionalization, NR of SAMs detected significant water penetration into each SAM composition, ranging from 1.6 to 5.7 water molecules per alkyl thiol when SAMs were immersed. This was the first direct measurement of water inside alkyl thiol SAMs and demonstrated that water accesses defects, amorphous regions, interdomain boundaries, and heterogeneous domains inherent to even well-formed SAMs. After peptide functionalization, water was found in both the SAM and peptide layers. The quantity of water in the SAM was twice that measured prior to peptide functionalization, suggesting the peptide disrupted the close-packed structure of the underlying SAM. To create an atomistic understanding of the amount of water measured around the peptide, we compared our NR data to previously published molecular dynamics (MD) simulations of the same peptide on a hydrophobic SAM in water and found the two techniques agreed. Combining these results with the dimensions of the peptide measured both experimentally and with MD, we hypothesize that immersing the peptide functionalized surface in water compressed the peptide closer to the SAM relative to its structure in ambient air. The amount of water readily accessible to the peptide-SAM interface reported here is the first step in assessing the quantity of water accessible to full proteins covalently bound to similar surfaces. Furthermore, we aim to test our protein immobilization hypothesis with azurin, an electron transfer protein, creating the groundwork for future immobilization experiments