Mechanisms for the enhanced thermal stability of a mutant of transcriptionfactor 1 as explained by H-1, N-15 and C-13 NMR chemical shifts and secondary structure analysis

A variant of the bacteriophage SPO1-encoded transcription factor 1 (TF1) wi th two site-specific mutations (E15G and T32I) was shown to be more thermal ly stable and bind DNA more tightly compared to the wild-type protein. In o rder 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 mo st of the backbone and sidechain sequential NMR assignments of the mutant p rotein, TF1-G15/I32. Insights derived from the H-1,N-15 and C-13 chemical s hifts and from the secondary structure analysis provide us with an explanat ion for the noted increase in thermal stability of TF1-G15/132. 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 i n the first two helices and their enclosed loop, Specifically, we have foun d that the second helix is extended by one residue at its N-terminus and ro tated 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 hydroph obic interactions between the flanking second and first helices and also be tween the second helix and the loop itself, Furthermore, the T32I mutation allows tighter packing between the second helix and the beta-sheet. Collect ively, these changes contribute to a more tightly associated dimer and henc e, to a greater thermal stability. (C) 2000 Elsevier Science B.V. All right s reserved.