APETHY: A general method for studying proteins bound to surfaces
Abstract
A general method has been developed for studying the attachment surface and conformational changes which accompany the binding of a protein to a macroscopic surface. This method is named APETHY, for Amide Proton Exchange by Tocsy and High-performance liquid chromatographY. The method involves packing the solid phase material into a column and attaching it to a conventional HPLC instrument. The protein is loaded onto the solid phase in aqueous solution, and a $\rm D\sb2O$ solution at high pH is pumped through the column to exchange exposed amide protons. The reaction is then quenched by pumping a low-pH $\rm D\sb2O$ solution over the column, and then the protein is eluted. After removing any eluant and concentrating the sample, the protein is redissolved in $\rm D\sb2O$ if necessary and a 2-D $\rm\sp1H$-$\sp1$ H TOCSY spectrum is acquired. Integration of the backbone NH-C$\alpha$H cross-peaks and comparison with appropriate control spectra then provides a quantifiable and highly reproducible measure of the degree of protection of each amide proton from exchange. In this way, the protein is able to "remember" its previous conformation long enough to be read in the NMR experiment. During the development of this technique, lysozyme has been observed to form five distinct layers on a DVB support at pH 7.4 in 10 mM aqueous sodium phosphate, and each can be collected and studied separately. A new method of presenting the amide-exchange protection data has also been developed, in which each observed amide proton is represented as its actual vector on a semi-transparent backbone, facilitating a clear and immediate comprehension of the pattern of protection and the visualization of the processes which led to it. Based on the use of the APETHY technique, a revised model for the binding of proteins to hydrophobic surfaces is proposed.
Degree
Ph.D.
Advisors
Gorenstein, Purdue University.
Subject Area
Biochemistry
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