Michael A. Cusanovich

Honors

• NIH Postdoctoral Fellowship, 1968-69
• NIH Career Development Award, 1975-80

Education and Appointments

• B.S. 1963, University of the Pacific
  
• Ph.D. 1967, UC San Diego
  

Research Summary

Energy transduction as mediated by proteins and enzymes plays a fundamental role in life processes. These range from respiration and photosynthesis to the visual process and phototaxis. My laboratory is engaged in the study of structure and function with proteins which participate in energy transduction. Our major focus is on cytochromes, although we have substantial experience with a variety of systems including: copper proteins, iron sulfur proteins, flavoproteins and enzymes, photosynthetic reaction centers and photoactive proteins. We are interested in a variety of issues related to the intrinsic properties of proteins which participate in electron transfer and photoactivation. For example, the physical and chemical interactions which mediate protein-protein interactions and as a consequence biological recognition and specificity, protein dynamics and stability, and the evolution of proteins and pathways. Our principal approach is to relate function to structure through the use of laser flash photolysis and/or stopped-flow spectroscopy to study function in real time (nanoseconds to seconds) and the use of NMR, X-ray crystallography, and a variety of spectroscopic techniques to understand the structure of proteins in both dynamic and static terms.

We currently have active projects in a number of areas:

1. Site directed mutagenesis of Rhodobacter capsulatus cytochrome c₂. The cytochromes c₂ are structural homologs of the mitochondrial cytochromes c but participate in photosynthetic electron transfer. Rb. capsulatus cytochrome c₂ is well characterized structurally and amenable to study in terms of interaction domains which mediate its reaction with electron donors and acceptors, protein stability, local domain dynamics, oxidation-reduction potentials, and heme electronic properties. The cytochrome c₂ system is particularly useful since mutants are prepared in Rb. capsulatus mutants where the wild-type cytochrome c₂ has been deleted. As a consequence, the in vivo function of the molecule can be related to physical-chemical alterations resulting from targeted mutations

2. PYP. Photoactive yellow protein (PYP) is a small, structurally well characterized protein which has a light initialed photocycle with features in common with the visual pigments and other photoactive proteins. In addition, PYP has been cloned, and site directed mutants are available. PYP offers the opportunity to obtain insights into basic information on protein-chromophore interactions critical for the function of visual pigments as well as light driven structural changes. Moreover, PYP has properties which make it a candidate for application in photobioelectronics and as a consequence is a potential biomaterial for the next generation of electronic devices.

3. Flavocytochromes c. The flavocytochromes c are a unique family of redox proteins which mediate electron flow between electron donors and acceptors through oriented intramolecular electron transfer involving multiple redox centers (heme and flavin). The flavocytochromes c have a number of unusual properties including the ability to interact with inorganic and organic sulfur containing compounds, for example, sulfide, sulfite, cysteine, and glutathione, through the formation of flavin adducts. A variety of studies are ongoing to elucidate the mechanism of action of the flavocytochromes c.

4. Cytochromes c₃. The cytochromes c₃ are tetraheme, low redox proteins which mediate electron transfer between hydrogen and other electron donors and sulfate. The cytochromes c₃ have the ability to self-organize in dry films and in these forms have electronic properties which may be useful in new electronic devices. As a result of the multiple hemes, much of the current work focuses on the flow of electrons within the cytochrome and the physical-chemical properties which control the properties of individual hemes.

Selected Publications

• “A sensitive circular dichroism marker for the chromophore environment of Photoactive Yellow Protein: assignment of the 307 and 318 bands to the n6 * transition of the carbonyl” B. Borucki, H. Otto, T. E. Meyer, M. A. Cusanovich and M. P. Heyn, J. Phys. Chem. B 109, 629-33 (2005).

• “Photoreversal kinetics of the I₁ and I₂ intermediates in the photocycle of Photoactive Yellow Protein by double flash experiments with variable time-delay” C.P. Joshi, B. Borucki, H. Otto, T. E. Meyer, M. A. Cusanovich and M. P. Heyn, Biochemistry 44, 656-65 (2005).

• “Thermochromatium tepidum Photoactive Yellow Protein/Bacteriophytochrome/Diguanylate Cyclase: Characterization of the PYP Domain” J. A. Kyndt, J. C. Fitch, T. E. Meyer and M. A. Cusanovich, Biochem. 44, 4755-64 (2005).

• “Structural evolution of the chromophore in the primary stages of trans/cis isomerization in photoactive yellow protein.” K. Heyne, O. F. Mohammed, A. Usman, J. Dreyer, E. T. Nibbering, and M. A. Cusanovich, J. Am. Chem. Soc. 127, 18100-6 (2005).

• “Time-Resolved Single Tryptophan Fluorescence in Photoactive Yellow Protein Monitors Changes in the Chromophore Structure during the Photocycle via Energy Transfer.” H. Otto, D. Hoersch, T. E. Meyer, M. A. Cusanovich, and M. P. Heyn, Biochem. 44, 16804-16 (2005).

• “Effect of Salt and pH on the Activation of Photoactive Yellow Protein and Gateway Mutants Y98Q and Y98F.” B. Borucki, J. A. Kyndt, C. P. Joshi, H. Otto, T. E. Meyer, M. A. Cusanovich, and M. P. Heyn, Biochem. 44, 13650-63 (2005)

• “GHP, a new c-type green heme protein from Halochromatium salexigens and other proteobacteria.” Gonzalez Van Driessche, Bart Devreese, John C. Fitch, Terrance E. Meyer, Michael A. Cusanovich, and Jozef J. Van Beeumen, FEBS J. 273, 2801-11 (2006).

• “Local Stability of Rodobacter capsulatus cytochrome c₂ Probed by Solution Phase Hydrogen/Deuterium Exchange and Mass Spectrometry” Guilong Cheng, Michael A. Cusanovich, Vicki H. Wysocki, J. Am. Soc. For Mass Spectrometry, in press.

• “Photocycle and Photoreversal of Photoactive Yellow Protein at Alkaline pH: Kinetics, Intermediates, and Equilibria. C.P. Joshi, B. Borucki, H. Otto, T.E. Meyer, M.A. Cusanovich, & M.P. Heyn. Biochem. 45, 7057-68 (2006).

• “The transient accumulation of the signaling state of photoactive yellow protein is controlled by the external pH” Berthold Borucki, Chandra P. Joshi, Harald Otto, Michael A. Cusanovich, and Maarten P. Heyn, Biophysical J., in press.

• “Properties of the Dark and Signaling States of Photoactive Yellow Protein Probed by Solution Phase Hydrogen/Deuterium Exchange and Mass Spectrometry” Guilong Cheng, Michael A. Cusanovich, Vicki H. Wysocki, Biochemistry, in press.