Synthetic model proteins: the relative contribution of leucine residues at the nonequivalent positions of the 3-4 hydrophobic repeat to the stability of the two-stranded alpha-helical coiled-coil.
Our de novo designed coiled-coil model protein consists of two identical 35-residue polypeptide chains arranged in a parallel and in-register alignment via interchain hydrophobic interactions and a disulfide bridge at the position 2 between two helices. To quantitate the relative contribution of leucine residues at the nonequivalent position of the 3-4 hydrophobic repeat to the stability of the two-stranded alpha-helical coiled-coil, a single alanine was systematically substituted for a leucine in each chain at position "a" (9, 16, 23, or 30) or "d" (5, 12, 19, 26, or 33). The formation and stability of the coiled-coils were determined by circular dichroism studies in the absence and presence of guanidine hydrochloride. All the proteins with an alanine substituted at position a have a similar stability ([Gdn.HCl]1/2 ranges from 2.6 to 2.9 M), while all the proteins with an alanine substituted at position d have similar stability ([Gdn.HCl]1/2 ranges from 3.6 to 4.2 M), except for the proteins with an alanine substituted in the C-terminal heptad. The greater decrease in stability observed for a Leu----Ala mutation at position a (the average delta delta Gu value is 3.3 kcal/mol) compared to those where the substitution was effected at position d (the average delta delta Gu value is 2.0 kcal/mol) indicates that an Ala mutation at position a has a greater effect on the side-chain packing and hydrophobic interactions in the coiled-coil than an Ala mutation at position d.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhou
N E
NE
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Kay
C M
CM
Hodges
R S
RS
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003198:Computer Simulation; D004220:Disulfides / Q000737:chemistry; D019791:Guanidine; D006146:Guanidines; D007930:Leucine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011506:Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D012091:Repetitive Sequences, Nucleic Acid; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D004220:Disulfides; D006146:Guanidines; D011506:Proteins; D007930:Leucine; D019791:Guanidine