Protein hydrogen exchange determined by mass spectrometry: A new tool for probing protein high-order structure and structural changes
Abstract
Protein amide hydrogen exchange has long been used as a sensitive probe of protein high-order structure and structural changes. The principal method for determining rates of hydrogen exchange in proteins has been multi-dimensional NMR. However, NMR is limited to the study of small and high soluble proteins. A new method, based on protein fragmentation and mass spectrometry to determine the rates of hydrogen/deuterium exchange in specific segments of a protein, was developed. In this method, the protein is fragmented by an acid protease after the protein undergoes H/D isotopic exchange. The amount of deuterium present in each specific fragment is determined by directly-coupled HPLC and mass spectrometry. Since all fast exchanging deuterons are washed out during HPLC fractionation, the deuterium contents determined by mass spectrometry are a direct measure of the number of deuterons in the backbone amide positions. Since the protein is digested into small peptides before the analysis, there is no limit in the size of the protein that can be studied. As a result, this method can be used to study spatially resolved structure and structural changes occurring in large proteins. The protein fragmentation/mass spectrometry method for determining structurally resolved protein hydrogen exchange rates was first developed using cytochrome c as a model. Heat induced unfolding of cytochrome c was also studied. The method was then used to study aldolase, a much larger protein (157 kDa). The hydrogen exchange results show great consistency to the three-dimensional structure of aldolase. The complexes of aldolase with the N-terminal peptide of band 3 and the cytoplasmic domain of band 3 were also studied. The results of this study show that there is a general loosening of aldolase structure in both complexes. This method is also useful for studies of protein unfolding. When this method was used to study the unfolding process of aldolase in different denaturing conditions, a detailed picture of the unfolding process was determined. When used to determine hydrogen exchange rates, mass spectrometry is becoming a very powerful tool in the study of protein high-order structure and structural changes.
Degree
Ph.D.
Advisors
Smith, Purdue University.
Subject Area
Biophysics|Biochemistry
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