Optimizing the Spin Labeling Reaction of Proteins for use in Protein-Protein and Protein-Heterochromatin Binding Characterization

Alexander Fuchs,  Chemistry

Faculty Mentor: Dr. Donald Hirsh

              Barrier-to-autointegration factor (BAF) is a protein found in the cell’s nucleus that, by binding to proteins in the nuclear membrane and DNA, assists in gene expression and in the integration of HIV into the host genome.  The goal of this research is to characterize binding between BAF and the LEM domain of Emerin, a protein of the inner nuclear membrane and binding between BAF and heterochromatin.  To do this, a stable radical or “spin-label” will be attached to BAF and its Electron Paramagnetic Resonance (EPR) spectrum will be recorded in the presence and absence of its binding partners.  S. cerevisiae iso-1-cytochrome c is relatively inexpensive and was used to optimize spin-labeling conditions, as described below.

              The spin-label MTSL forms a disulfide bond to cysteine residues of peptides.  The concentration of iso-1-cytochrome c was quantified using its known molar absorptivity at 410 nm.  The concentration of spin-label in the same solution was determined by EPR spectroscopy.  A standard curve of the EPR signal v. concentration was made using TEMPOL, an inexpensive spin-label.  EPR spectra collected from spin-labeled protein were then used to quantify the concentration of spin-label.  Approximately 100% of iso-1-cytochrome c was spin-labeled using 1 mM DTT reductant and 5.62 mM MTSL solution.  Studies with Human BAF have been initiated, but mutants with accessible cysteine residues are not yet available.

Personal Statement

              The SURP has provided me with an excellent academic environment for independent biochemical research with assistance from and collaboration with students and faculty in both the biological and chemical fields.  I have gained exposure to instruments such as the EPR instrument that I would not otherwise have access to at this point in my studies.