Date of Award

6-2001

Document Type

Open Access

Degree Name

Bachelor of Science

Department

Chemistry

Language

English

Abstract

Homeodomain proteins are transcription factors that contain a conserved 60- residue sequence, beginning with an N-terminal unstructured arm, followed by an alpha helix, a loop, and a helix-tum-helix. The yeast protein MATa1 is unusual among homeodomains in that, as a monomer, it binds very poorly to its DNA operator. However, the a1- α2 heterodimer binds to the hsg operator with 3000 times the affinity it has for nonspecific DNA. Studies have shown that most of the heterodimer's binding specificity is due to a1 rather than α2 (1,2). To identify the structural changes that transform al into a strong, sequence-specific DNA binding protein, a single-point mutant (s25y) and a double-point mutant (q24r/s25y) were studied. EMSA studies showed that both mutants bind to DNA with greater affinity than wild type al does. Analysis of 2-D 15N-HSQC and 3-D 15N-NOESY spectra showed that significant changes in the chemical shifts of the backbone amide groups of loop 1 and helix 3 occur upon mutation. The NOESY spectrum was also used to identify NOEs between amide protons of sequential residues, indicating where alpha helical conformations occurred. The NOEs showed that the third helix is extended in the a1 mutants. Finally, titration experiments were performed by adding aliquots of the 19- residue a2 tail peptide to al and to each al mutant, and then recording HSQC spectra. These showed that chemical shift changes which occur in wild type al upon a2 tail binding are diminished in the al mutants.

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