The Application of Single-Pass Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy for Protein Analysis

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Title: The Application of Single-Pass Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy for Protein Analysis
Author: Smith, Brandye Michelle
Advisors: Daniel L. Feldheim, Committee Member
Kenneth W. Hanck, Committee Member
Paul Wollenzien, Committee Member
Stefan Franzen, Committee Chair
Abstract: Application of single-pass attenuated total reflection Fourier-transform infrared (ATR-FT-IR) spectroscopy was investigated for the following; 1) secondary structure protein analysis, 2) protein secondary structure prediction, 3) amino acid classification, 4) peptide conformational, and 5) protein binding interactions. This research is the first reported application of single-pass ATR-FT-IR for protein analysis thus the method was validated using transmission FT-IR and multi-pass ATR-FT-IR as referee methods. The technique was advantageous since it permitted rapid secondary structure analysis on small volumes of protein in H2O solution without the use of demountable thin pathlength sample cells and without performing a H2O subtraction. Principle component regression (PCR) was applied to a spectral library of proteins in H2O solution acquired by single-pass ATR-FT-IR spectroscopy to predict the secondary structure content, principally a-helical and the b-sheet content, of proteins within a spectral library. An 'inside model space' bootstrap and a genetic algorithm (GA) were successfully used to improve prediction results. Three spectral libraries are presented and the validation results yielded an average absolute error of 1.6% for a-helix content prediction and an average absolute error of 1.7% for b-sheet content prediction. Single-pass ATR-FT-IR and normal mode analysis was employed to study amino acids and their role in the spectra of peptides. An amino acid identification model based upon Mahalanobis' distance yielded 100% correct classification for all 20 amino acids used in this study. Comparisons of peptides in free solution and those associated with proteins were made and it was established that the single-pass ATR method has a sufficient signal-to-noise ratio to obtain difference spectra for peptides in solution and peptides conjugated or bound to proteins. This study showed that protein-associated peptides differ in conformation from those in free solution. To investigate protein-binding interactions a Ge internal reflectance element was modified. Successful binding of his-tagged dehaloperoxidase (DHP) and his-tagged biotin via a nickel nitrilotriacetic acid (Ni-NTA) surface was monitored via single-pass ATR-FT-IR. The surface modification method presented had minimal non-specific binding, the surface was re-usable, and removal of the organic surface was achievable.
Date: 2003-04-07
Degree: PhD
Discipline: Chemistry

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