DEPARTMENT of PHYSIOLOGY

Other Perelman School of Medicine Affiliations

• Cell and Molecular Biology Graduate Program

• Program in Cellular Physiology

Degrees & Education

• BA, Brandeis University, 1966

• MPhil, Yale University, 1969

• PhD, Yale University, 1972

Awards & Honors

• 2003-2007 NIH Study Section (MDCN-3)

• 2003-2004 President, Society of General Physiologists

• 2001-2002 Present Editorial Board, Journal of General Physiology

• 1996-1998 Society of General Physiologists Counci

• 1990 Lindback Distinguished Teaching Award, University of Pennsylvania

• 1985-1988 NSF Cell Physiology Advisory Panel

• 1981-1986 Research Career Development Award

Professional Affiliations

• Society of General Physiologists

• Biophysical Society

Research Description

Our program aims to elucidate fundamental principles of protein folding in the formation of a voltage-gated potassium (Kv) channel, starting with its inception at the ribosome where Kv mRNA is translated and proceeding to the sequence of events underlying its integration into the bilayer of the endoplasmic reticulum. Knowledge of the mature Kv channel in the plasma membrane cannot tell us about the channel’s history of distinct biogenic stages; thus we focus on early folding events of the Kv protein. The stepwise progression of Kv channel formation is governed by a dynamic interaction between Kv protein, the ribosomal exit tunnel, and the translocon. The latter two are participatory conduits for nascent peptides during their synthesis and membrane integration. We use novel applications of our recently developed folding and accessibility assays, new unnatural amino acids/synthetase inhibitors, real-time translation kinetics, electrophysiology of Xenopus oocytes, photocrosslinking in the ribosomal tunnel and translocon, and computational approaches to probe the three major domains of a Kv channel (T1, voltage-sensor, and pore) and to define peptide-tunnel interactions for a broad range of peptides. Our findings will be particularly relevant to understanding defects in protein biogenesis and potential therapeutic strategies at the ribosome.

Data Protocols & Available Constructs

• Tape Measure Constructs:

• Extended tape measure nascent chains (single-cysteine mutants)

• Poly-Arg constructs with T1 Kv1.3 N-terminus (WT and mutants)

• Extended nascent chain with cysteine reporter and charged amino acids at the ribosome constriction

• Poly-Ala constructs in an extended tape measure

• Tandem tape measure constructs with/without poly-Ala tracts

• Extended tape measures with folded N-terminal domains

• Extended tape measures with truncated T1 domains

• Poly-Ser constructs in an extended tape measure




• Secondary Folding Assay:

• Pegylation Protocols and cysteine-scanning constructs

• Protocols using inserted poly-Ala tracks




• Tertiary Folding Assay:

• Pfold determination using bifunctional maleimides and PEG-MAL/PEG-SH

• Modification kinetics using pegylation (kmod)




• Location in the ribosomal exit tunnel:

• Pegylation accessibility assay

• Photocrosslinking using nitrene-generator

• TMA modification kinetics (kmod)




• Electrostatic Potential Determinations:

• Constructs with reporter cysteines, poly-Arg and poly-Asp tracts




• Unnatural Amino Acid Incorporation:

• Flexizyme protocols for aminoacylation of tRNAs

• Synthetase inhibition protocols

• Rescue of arrested peptides at Cysteine or Amber codons