Shape and energetics of a cavity in c-Myb probed by natural and non-natural amino acid mutations.
The shape and the energetics of a functional cavity in the R2 subdomain (90-141) of the c-Myb DNA-binding domain were investigated by spectroscopy and thermodynamic analysis. We focused on the valine 103 residue located in front of the cavity. Nine mutants, in which valine 103 was substituted with alanine, 2-aminobutyric acid, norvaline, norleucine, leucine, isoleucine, allo -isoleucine, cyclohexylglycine, and cyclohexylalanine, were chemically synthesized and analyzed. These mutants provided a wide distribution of sizes which ranged from forming additional cavity space to filling and overflowing the cavity space. Temperature-scanning circular dichroism measurements and differential scanning calorimetry revealed a linear relationship between the van't Hoff enthalpy and the thermal transition temperature for the cavity-filling mutations. On the other hand, the mutants with side-chains larger than the side-chain of leucine resulted in a relatively low transition enthalpy and temperature, most likely due to the exposure of the side-chain to solvent and the increase in the entropy of the folded states. Branching at the beta-carbon atom reduced the unfolding free energy due to the steric constraint in the cavity. In particular, the mutational elongation of the side-chain from beta-carbon to the trans -to-CO direction proved to be more hindered than that from beta-carbon to the trans -to-NH. The unfolding free energy versus side-chain volume formed a bell-shaped plot with a maximum free energy for the leucine mutant. The difference in the transition free energy for cavity-filling mutants with beta-unbranched side-chains were two to four times larger than the difference in the transfer energy from organic solvent to water. Therefore, the increase in unfolding free energy would most likely be attributed to van der Waals interactions in the cavity wall, which would be a origin of stabilization by the sliding of tryptophan 95 into the cavity upon DNA binding.
Morii
H
H
National Institute of Bioscience and Human-Technology (NIBH), Tsukuba, Ibaraki, 305-8566, Japan. morii@nibh.go.jp
Uedaira
H
H
Ogata
K
K
Ishii
S
S
Sarai
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D020598:Proto-Oncogene Proteins c-myb / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D014633:Valine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D020598:Proto-Oncogene Proteins c-myb; D014364:Tryptophan; D014633:Valine