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Department of Structural Biology (412); 383-7325 Research Overview. My laboratory's main research interest is in membrane receptors. We try to address questions related to membrane receptors from a molecular and structural perspective using an interdisciplinary combination of molecular biology, biochemistry, biophysics, structural biology and computational biology. We are particularly interested in membrane receptor folding/misfolding, their conformational changes and their interactions. Rhodopsin and retinal degeneration. Our favorite receptor is the G protein coupled receptor rhodopsin, the visual pigment in the eye. Rhodopsin is one of the genetic causes for a retinal degeneration disease, Retinitis pigmentosa. Misfolding of rhodopsin is a demonstrated cause for the disease, but it has been difficult to explain the variability in symptoms and onset of the disease seen in patients with different mutations. We are therefore investigating in depth the effects of mutations seen in Retinitis pigmentosa families on folding, dynamics, structure and functions of the purified rhodopsin molecules. Structure and conformational changes in membrane receptors. To improve our understanding of this topic we apply biochemical (exploiting cysteine mutagenesis and chemistry);, biophysical (NMR, CD, fluorescence and in collaboration EPR, X-ray and small angle neutron scattering); and computational (machine learning and structural analysis); techniques to not only rhodopsin, but also other mammalian membrane receptors, in particular the metabotropic glutamate receptors, epidermal growth factor receptor, growth hormone receptor and the archabacterial phototaxis transducer, that has homology to bacterial chemotaxis receptors. Folding of membrane receptors. Our studies of folding/misfolding in rhodopsin have revealed that the current paradigm for membrane protein folding, the two-stage hypothesis based on bacteriorhodopsin studies, is not sufficient to explain rhodopsin folding and stability. This has led to a new hypothesis for membrane protein folding, the long-range interactions model, that is more in line with current thinking for the folding of soluble proteins. Development of new methods. We have developed new computational approaches for prediction of protein characteristics related to protein structure, dynamics, interactions and function in an NSF-funded large-scale collaboration between experimentalists and computer scientists. For these methods, we have shown general applicability beyond the specific systems that we study experimentally. My laboratory also develops new experimental approaches, including 19F-NMR based approaches to membrane protein structural studies in solution. New research initiatives: Molecular basis of vision and visual impairment. We are actively seeking new hypotheses as to the effect of rhodopsin mutations on the retina. We have also started new collaborations with the Ophthalmology department on the study of the molecular basis for glaucoma and age-related macular degeneration, starting with the unified general hypothesis that misfolding is a contributor in both disease groups. We are also investigating other retinal proteins: (i); metabotropic glutamate receptor type 6, an ON-bipolar cell-specific glutamate-binding G protein coupled receptor and (ii); glutamic acid rich proteins (GARP's), highly negatively charged retina-specific proteins with likely Calcium-buffering function. We have recently begun to study the molecular basis of vision enhancement by anthocyanins and porphyrins. Viral membrane proteins and virus-host cell receptor interactions. We are applying our prediction algorithms to HIV and SIV proteins, with particular interest in the gp41 membrane protein and its interactions with gp120. We are conducting biophysical and structural studies, in collaboration with researchers at the University of Pittsburgh who study HIV function. Interactions of membrane receptors. We have been working on predicting protein protein interactions by integrating diverse biological evidences using machine learning methods for several years, starting with the model organism yeast. We have now moved to the stage where we can predict human interactions, which has recently generated many novel binding partners for membrane receptors, that we verify experimentally for rhodopsin and the epidermal growth factor receptor. To complement our interactions studies with functional significance, we have initiated several collaborations with biomedical scientists at the University of Pittsburgh. One particularly exciting avenue is our recent prediction that rhodopsin may interact with chemokines, which is not only interesting from a rhodopsin perspective but also from a chemokine receptor perspective. Publications 1. Budyak, I., Pipich, V., Mironova, O., Schlesinger, R., Zaccai, G. & Klein-Seetharaman, J. (2006) Shape and oligomerization state of the cytoplasmic domain of the phototaxis transducer II from Natronobacterium pharaonis. Proc. Natl. Acad. Sci. USA 103(42), 15428-33. 2. Ganapathiraju, M., Balakrishnan, N., Reddy, R. and Klein-Seetharaman, J. (2007) Transmembrane helix prediction using latent semantic analysis, BMC Bioinformatics (Incob Proceedings), in press. 3. Iannaccone, A., Man, D., Waseem, N., Jennings, B.J., Ganapathiraju, M., Gallaher, K., Reese, E., Bhattacharya, S.S. and Klein-Seetharaman, J. (2006) Retinitis Pigmentosa Associated with Rhodopsin Mutations: Correlation between Phenotypic Variability and Molecular Effects. Vision Research 46(27), 4556-67. 4. Tastan, O., Yu, E., Ganapathiraju, M., Aref, A., Rader, A.J. and Klein-Seetharaman, J. (2007) Comparison of stability predictions and simulated unfolding of rhodopsin structures. Photochemistry and Photobiology, in press. 5. Karla Werner, Ines Lehner, Harpreet Kaur Dhiman, Christian Richter, Clemens Glaubitz, Harald Schwalbe, Judith Klein-Seetharaman and H. Gobind Khorana (2007) Combined solid state and solution NMR studies of α,ε-15N labeled bovine rhodopsin. J. Biomol. NMR 37(4), 303-312. 6. Qi, Y., Bar-Joseph, Z. and Klein-Seetharaman, J. (2006) Evaluation of different biological data and computational classification methods for use in protein interaction prediction. Proteins - Structure, Function and Bioinformatics 63, 490-500. 7. Klein-Seetharaman, J. (2005) Dual role for long-range interactions in helical membrane receptor folding and signaling. Trends in Pharmacological Sciences 26, 183-189. People in the lab |
  
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Upcoming events
- Seminar: "Structure-based design of organometallic kinase inhibitors", Ronen Marmorstein, Ph.D. (event)(10 days)
- Seminar - Adam Linstedt, Ph.D.(event)(17 days)
- Seminar - Chunyu Wang, Ph.D. (event)(52 days)
