Katherine Kellersberger University of Maryland, Baltimore County Office: 410-455-1406 FAX: 410-455-2608 E-mail: kakeller@umbc.edu Job Title: Research Associate Ph.D. in Analytical Chemistry from Brigham Young University

Speaker: Katherine Kellersburger, University of Maryland, Baltimore County, Baltimore, MD 21250

Topic: The Zinc-finger Protein HIV-1 Nucleocapsid p7 Studied by Electron Capture Dissociation ESI-FTMS - Video (running time 00:37:03) ^*

Place: Building 549, Auditorium, NCI at Frederick, Frederick, MD

Time: Tuesday, November 18, 2003, at 2:00 PM

Abstract: HIV-1 nucleocapsid protein p7 (NC) is a zinc-finger protein that plays crucial roles in genome recognition, dimerization, and packaging of viral RNA into infectious particles. Much of the functionality of NC is believed to arise from the specific architecture of its two zinc-finger domains, which are highly conserved regions found in all known retroviruses. Prior studies have shown that mutation of NC zinc-finger domains renders the viral particle non-infectious. Therefore, complete elucidation of the chaperone properties and RNA-binding mechanism of NC are necessary for the development of small drug inhibitors aimed at disrupting the packaging of the AIDS virus.

Electrospray ionization in combination with Fourier transform mass spectrometry (ESI-FTMS) is rapidly becoming the method of choice for the characterization of proteins and protein-nucleic acid complexes. A variety of gas-phase fragmentation techniques, such as collision-induced dissociation (CID), have long been used to study protein sequence and structure via mass spectrometry. However, even the relatively low-energy CID afforded by ICR-based methods has a tendency to both disrupt tertiary structure and cause loss of post-translational modifications (PTMs). Consistent with this observation, SORI-CID experiments performed by our group on holo-NC have shown that Zn is ejected from the protein structure prior to fragmentation of the amino acid backbone.

The recent development of electron-capture dissociation (ECD)1 has shown great promise in the investigation of non-covalent complexes, largely due to the non-ergodic nature of the fragmentation which provides unique sequence information with minimal loss of PTMs. In fact, the fragmentation of larger proteins with considerable tertiary structure often requires additional energy in the form of collision gas, heat, or longer hexapole storage times to disrupt intramolecular bonds2. Despite the growing number of applications involving the use of ECD, little is known about its effects on metal chelation by metalloproteins. For this reason, we have undertaken the study of NC using ESI-FTMS combined with ECD to determine whether this is a viable method for the characterization and structural elucidation of metalloproteins, and, eventually, protein-nucleic acid complexes. When applied to the analysis of both apo- and holo-NC, distinct differences were observed in the both the pattern and frequency of ECD-induced fragmentation. Furthermore, ECD of the individual zinc-finger domains F1 and F2 showed a high incidence of metal retention by the fragment ions, with many fewer zinc-retaining fragments present following fragmentation of F2 than F1. These results present an interesting correlation with solution-phase data, which indicate that zinc is ejected from F2 more easily than from F1. Although far from conclusive, these data suggest that there may be a "minimum structure" required for the retention of Zn ion, and that ECD may constitute a viable method for the characterization of metalloproteins, and, eventually, protein-nucleic acid complexes.

The slides to this seminar are in a 2.2 Megabyte PDF file, which can be opened and read by using the free Adobe Acrobat Reader®.

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