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

Diversity & Inclusion Initiatives

• Founding and continuing member of Department of Physiology Diversity, Equity, Inclusion Committee and attended all of Diversity, Equity, Inclusion committee functions.
• Member of the Cell Biology, Physiology and Metabolism (CPM) graduate group and interview graduate applicants, including recruitment of diversity trainees.