Probing the methyl donor, S-adenosyl-L-methionine, binding site of the Ste14p isoprenylcysteine carboxyl methyltransferase using electron paramagnetic resonance (EPR) spectroscopy

Ulhas Sopanrao Kadam, Purdue University

Abstract

The isoprenylcysteine carboxyl methyltransferase (Icmt) family of enzymes performs the methylation step of the –CaaX motif post-translational processing pathway. Ste14p from S. cerevisiae is the founding member of the Icmt family and is responsible for the methyl group transfer from S-adenosyl-L-methionine (SAM) to numerous methyl accepting substrates in yeast. Ste14p, which is localized to the membrane of the endoplasmic reticulum, is a 26-kDa integral membrane protein and possesses six transmembrane spanning segments. Sequence analysis and homology studies reveal a putative SAM binding region at the C-terminus region, but it is not known which residues are important for SAM binding and catalysis. This study focused on elucidating which regions of Ste14p may be involved in SAM binding using site-directed spin labeling (SDSL) coupled with electron paramagnetic resonance (EPR) spectroscopy. The study found that His-Ste14p changed conformation upon the addition of 5 mM SAM when residues S222C and E227C were spin-labeled with MTSL. Furthermore, the study explored the interaction of JM-4-68B, an inhibitor of Ste14p, with the protein. The residues S222C and A223C showed conformational changes upon incubation with the Ste14p inhibitor JM-4-68B, His-Ste14p spin labeled on residues S222C and A223C also demonstrated a conformational change. Together, these data corroborate the hypothesis that the C-terminus region is important for biological activity and SAM binding in Ste14p. Determination of critical residues in SAM binding in Ste14p will give an insight into the mechanism of action of the Icmt class of enzymes.

Degree

M.S.

Advisors

Hrycyna, Purdue University.

Subject Area

Molecular biology|Biochemistry|Biophysics

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