The aim of this project is to develop and test for feasibility combined protein engineering and isotope edited Fourier transform infrared (FTIR) techniques to analyze the molecular mechanisms of membrane translocation by protein toxins. The focus will be on diphtheria toxin (DT), an AB toxin that contains the receptor binding (R), transmembrane (T), and catalytic (C) domains. The crossing of the endosome membrane by DT during cytosol translocation is facilitated by its T-domain. However, the underlying structural mechanisms, i.e. the conformational transitions in the T-domain that mediate membrane pore formation, are not well understood. The overall hypothesis of this proposal is that major conformational changes occur in the T-domain of DT upon pore formation and membrane translocation, which will be identified by innovative biophysical approaches. Selected subunits or segments of the protein will be labeled with the 13C stable isotope using native and kinetically controlled peptide ligation techniques. Polarized FTIR spectroscopy will be used to identify site- specific conformational and orientational changes in DT during membrane pore formation and protein translocation. Real-time structural changes during membrane insertion of the protein will be monitored by stopped-flow fluorescence and time-resolved FTIR spectroscopy. Structural analysis of subunit- or segment- selective isotope labeled proteins by vibrational spectroscopy is a powerful technique that has not been utilized thus far. Development of this technique will help gain insight in site-resolved structural changes in proteins that underlie defined functions. It is anticipated that labeling of whole subunits or larg segments within a protein with stable isotopes and structural analysis by FTIR will be accomplished within this project. The following Specific Aims will be pursued.
Specific Aim 1. Produce subunit-specific stable isotope-labeled diphtheria toxin (DT) and segmentally labeled T-domain of DT for structural studies. Recombinant or semisynthetic proteins will be produced in which a whole subunit or a segment is labeled with the stable isotope 13C. First, the uniformly 13C-labeled catalytic domain of DT will be expressed in E. coli and disulfide bridged to the unlabeled B-chain or the T-domain. Second, several constructs of the T-domain in which defined segments are selectively 13C-labeled will be produced by peptide ligation techniques. This approach will allow FTIR spectral resolution and identification of site-specific conformational and orientational changes in proteins that facilitate membrane pore formation and protein translocation.
Specific Aim 2. Identify the dynamic conformational/orientational changes in diphtheria toxin and its T domain that underlie membrane translocation. The hypothesis that DT T-domain undergoes major conformational/orientational changes during pore formation will be tested by FTIR studies on the subunit- and segmental- 13C-labeled protein. Time- dependent FTIR measurements will reveal changes in the secondary structure and the orientation of defined regions of the protein during membrane insertion and translocation. Conformational changes in both the 13C- labeled C-domain and unlabeled T-domains of DT during membrane translocation will be identified. Time- resolved fluorescence studies will reveal dynamic changes in the tertiary structure and the kinetics of membrane insertion.

Public Health Relevance

Protein toxins need to cross one or more cellular membranes to enter the cell and damage or kill it. The mechanisms of membrane translocation are not well understood and require further studies. The aim of this project is to develop and test for feasibility advanced protein engineering and biophysical approaches to analyze the molecular mechanisms of membrane translocation by protein toxins, such as diphtheria toxin. Better understanding of the molecular mechanisms of protein toxins will facilitate development of novel anti- toxin therapies.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Small Research Grants (R03)
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Membrane Biology and Protein Processing (MBPP)
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Taylor, Christopher E,
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University of Central Florida
Schools of Arts and Sciences
United States
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