Current research in the Knight laboratory focuses on molecular interactions of proteins and lipid membranes, and on the development of single-molecule techniques for measuring them. Membrane-targeting proteins are normally soluble in the cytoplasm, but are recruited to membrane surfaces during cell signaling events. These proteins typically dock to the lipid membrane through one or more different types of molecular interactions, including (1) specific recognition of lipid headgroups as ligands; (2) nonspecific electrostatic interaction of positively charged regions of the protein with negatively charged lipids; (3) insertion of nonpolar amino acid sidechains into the hydrophobic interior of the membrane; and/or (4) interaction with other proteins on the membrane surface. We are particularly interested in discovering the molecular mechanisms of membrane interaction for proteins involved in insulin secretion. These interactions are expected to reveal important features of the proteins and membranes that enable proper regulation of secretion, and may offer clues to how secretion is dysregulated during progression of type II diabetes.
Biochemical and biophysical techniques are being used to investigate a number of individual membrane-targeting protein domains. These include a protein-to-membrane fluorescence resonance energy transfer method that can be used to measure both equilibria and kinetics of membrane binding. In addition, Dr. Knight’s recent and ongoing research has developed new applications of single-molecule fluorescence microscopy for measuring the kinetics and stoichiometry of protein-membrane binding.
Dr. Knight was introduced to the field of biophysical chemistry as an undergraduate chemistry major in the lab of Dorothy Erie. His undergraduate research involved RNA polymerases from thermophilic bacteria. Ph.D. research was completed in the lab of Prof. Andrew Miranker and focused on membrane interactions and amyloid aggregation of islet amyloid polypeptide (IAPP), a peptide secreted with insulin that forms insoluble aggregates in the diabetic pancreas. Dr. Knight continued to pursue interests in protein-membrane interaction through postdoctoral research in the laboratory of Prof. Joseph Falke at the University of Colorado at Boulder. There, his research developed methods using total internal reflection fluorescence microscopy to study these interactions at the single-molecule level.
Ph.D., Pharmacology, Yale University, 2006
B.S., Chemistry, University of North Carolina, 2000
Refereed research articles (supervised students italicized)
Vasquez, J.K., Chantranuvatana, K., Giardina, D.T., Coffman, M.C., and Knight, J.D. (2014) Lateral diffusion of proteins on supported lipid bilayers: Additive friction of synaptotagmin 7 C2A-C2B tandem domains. Biochemistry 53, 7904–7913. http://pubs.acs.org/doi/abs/10.1021/bi5012223
Lyakhova, T.A. and Knight, J.D. (2014) The C2 domains of granuphilin are high-affinity sensors for plasma membrane lipids. Chemistry and Physics of Lipids, 182, 29-37. Invited submission for special issue on phosphoinositides. http://www.sciencedirect.com/science/article/pii/S0009308413001370
Ziemba, B.P., Li, J., Landgraf, K.E., Knight, J.D., Voth, G.A., Falke, J.J. (2014) Single-molecule studies reveal a hidden key step in the activation mechanism of membrane-bound protein kinase C-α. Biochemistry 53, 1697-1713. http://pubs.acs.org/doi/abs/10.1021/bi4016082
Yamamoto, T.M., Cook, J.M., Kotter, C.V., Khat, T., Silva, K.D., Ferreyros, M., Holt, J.W., Knight, J.D., and Charlesworth, A. (2013) Zar1 represses translation in Xenopus oocytes and binds to the TCS in maternal mRNAs with different characteristics than Zar2. Biochimica et Biophysica Acta – Gene Regulatory Mechanisms, 1829, 1034-1046.
Brandt, D.S., Coffman, M., Falke, J.J., and Knight, J.D. (2012) Hydrophobic contributions to the membrane docking of synaptotagmin 7 C2A domain: Mechanistic contrast between isoforms 1 and 7. Biochemistry 51, 7654-64.
Ziemba, B.P., Knight, J.D., Falke, J.J. (2012) Assembly of Membrane-Bound Protein Complexes: Detection and Analysis by Single Molecule Diffusion. Biochemistry 51(8):1638-47.
Knight, J.D., Lerner, M.G., Marcano-Velázquez, J.G., Pastor, R.W., and Falke, J.J. (2010) Single molecule diffusion of membrane-bound proteins: Window into lipid contacts and bilayer dynamics. Biophysical Journal 99, 2879-87.
Knight, J.D. and Falke, J.J. (2009) Single-molecule fluorescence studies of a PH domain: new insights into the membrane docking reaction. Biophysical Journal 96, 566-82.
Knight, J.D., Williamson, J.A., and Miranker, A.D. (2008) Interaction of membrane-bound islet amyloid polypeptide with soluble and crystalline insulin. Protein Science 17, 1850-56.
Knight, J.D., Hebda, J.A., and Miranker, A.D. (2006) Conserved and cooperative assembly of membrane-bound a-helical states of islet amyloid polypeptide. Biochemistry 45, 9496-9508.
Knight, J.D. and Miranker, A.D. (2004) Phospholipid catalysis of diabetic amyloid assembly. Journal of Molecular Biology 341, 1175-1187.
Knight, J.D. and Adami, R.C. (2003) Stabilization of DNA utilizing divalent cations and alcohol. International Journal of Pharmaceutics 264,15-24.
Eakin, C.M., Knight, J.D., Morgan, C.J., Gelfand, M.A., Miranker, A.D. (2002) Formation of a copper specific binding site in non-native states of b-2-microglobulin. Biochemistry 41, 10646-56.
Biochemistry (CHEM 3810)
Biochemistry Laboratory (CHEM 4828)
General Biochemistry I (CHEM 4810/5810)
General Biochemistry II (CHEM 4820/5820)
Physical Biochemistry Laboratory (CHEM 4518)