title: Stabilizing and destabilizing clusters in the hydrophobic core of long two-stranded alpha-helical coiled-coils.
abstract: Detailed sequence analyses of the hydrophobic core residues of two long two-stranded alpha-helical coiled-coils that differ dramatically in sequence, function, and length were performed (tropomyosin of 284 residues and the coiled-coil domain of the myosin rod of 1086 residues). Three types of regions were present in the hydrophobic core of both proteins: stabilizing clusters and destabilizing clusters, defined as three or more consecutive core residues of either stabilizing (Leu, Ile, Val, Met, Phe, and Tyr) or destabilizing (Gly, Ala, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Arg, Lys, and Trp) residues, and intervening regions that consist of both stabilizing and destabilizing residues in the hydrophobic core but no clusters. Subsequently, we designed a series of two-stranded coiled-coils to determine what defines a destabilizing cluster and varied the length of the destabilizing cluster from 3 to 7 residues to determine the length effect of the destabilizing cluster on protein stability. The results showed a dramatic destabilization, caused by a single Leu to Ala substitution, on formation of a 3-residue destabilizing cluster (DeltaT(m) of 17-21 degrees C) regardless of the stability of the coiled-coil. Any further substitution of Leu to Ala that increased the size of the destabilizing cluster to 5 or 7 hydrophobic core residues in length had little effect on stability (DeltaT(m) of 1.4-2.8 degrees C). These results suggested that the contribution of Leu to protein stability is context-dependent on whether the hydrophobe is in a stabilizing cluster or its proximity to neighboring destabilizing and stabilizing clusters.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
authors.forename: Stanley C
authors.initials: SC
authors.lastname: Kwok
authors.forename: Robert S
authors.initials: RS
authors.lastname: Hodges
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004355:Drug Stability; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009218:Myosins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011506:Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism
chemical_list: D010455:Peptides; D011506:Proteins; D014335:Tropomyosin; D009218:Myosins