University of Colorado Denver College of Liberal Arts and Sciences

Bio

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.

Select Publications

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." Biophys J, in press.

Knight, J.D. and Falke, J.J. (2009) "Single-molecule fluorescence studies of a PH domain: new insights into the membrane docking reaction." Biophys J 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 α-helical states of islet amyloid polypeptide." Biochemistry 45, 9496-9508.

Knight, J.D. and Miranker, A.D. (2004) "Phospholipid catalysis of diabetic amyloid assembly." J Mol Biol 341, 1175-1187.

Knight, J.D. and Adami, R.C. (2003) "Stabilization of DNA utilizing divalent cations and alcohol." Int J Pharm 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 β-2-microglobulin." Biochemistry 41, 10646-56.

Courses Taught

CHEM 4810/5810: General Biochemistry I
CHEM 4820/5820: General Biochemistry II