Mechanisms for the enhanced thermal stability of a mutant of transcription factor 1 as explained by (1)H, (15)N and (13)C NMR chemical shifts and secondary structure analysis.
A variant of the bacteriophage SPO1-encoded transcription factor 1 (TF1) with two site-specific mutations (E15G and T32I) was shown to be more thermally stable and bind DNA more tightly compared to the wild-type protein. In order to understand the biochemical mechanisms underlying these properties, we are engaged in determining the solution structures of this mutant alone and in complex with DNA using nuclear magnetic resonance (NMR) spectroscopy. The first phase of this project is reported here, as we have completed most of the backbone and sidechain sequential NMR assignments of the mutant protein, TF1-G15/I32. Insights derived from the (1)H, (15)N and (13)C chemical shifts and from the secondary structure analysis provide us with an explanation for the noted increase in thermal stability of TF1-G15/I32. Compared to the structure of the wild-type protein, the beta-sheet and the C-terminal helix remain largely unaffected whereas the mutations cause great changes in the first two helices and their enclosed loop. Specifically, we have found that the second helix is extended by one residue at its N-terminus and rotated in a way that allows Ala-37 to interact with Tyr-94 of the C-terminal helix. The loop has been found to become more rigid as a result of hydrophobic interactions between the flanking second and first helices and also between the second helix and the loop itself. Furthermore, the T32I mutation allows tighter packing between the second helix and the beta-sheet. Collectively, these changes contribute to a more tightly associated dimer and hence, to a greater thermal stability.
Vu
H M
HM
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA.
Liu
W
W
Grove
A
A
Geiduschek
E P
EP
Kearns
D R
DR
D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004355:Drug Stability; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009154:Mutation; D017433:Protein Structure, Secondary; D014764:Viral Proteins
D004268:DNA-Binding Proteins; C059361:TF1 DNA-binding protein, Bacteriophage SPO1; D014764:Viral Proteins