Identification of a unique "stability control region" that controls protein stability of tropomyosin: A two-stranded alpha-helical coiled-coil.
Nine recombinant chicken skeletal alpha-tropomyosin proteins were prepared, eight C-terminal deletion constructs and the full length protein (1-81, 1-92, 1-99, 1-105, 1-110, 1-119, 1-131, 1-260 and 1-284) and characterized by circular dichroism spectroscopy and analytical ultracentrifugation. We identified for the first time, a stability control region between residues 97 and 118. Fragments of tropomyosin lacking this region (1-81, 1-92, and 1-99) still fold into two-stranded alpha-helical coiled-coils but are significantly less stable (T(m) between 26-28.5 degrees C) than longer fragments containing this region (1-119, 1-131, 1-260 and 1-284) which show a large increase in their thermal midpoints (T(m) 40-43 degrees C) for a DeltaT(m) of 16-18 degrees C between 1-99 and 1-119. We further investigated two additional fragments that ended between residues 99 and 119, that is fragments 1-105 and 1-110. These fragments were more stable than 1-99 and less stable than 1-119, and showed that there were three separate sites that synergistically contribute to the large jump in protein stability (electrostatic clusters 97-104 and 112-118, and a hydrophobic interaction from Leu 110). All the residues involved in these stabilizing interactions are located outside the hydrophobic core a and d positions that have been shown to be the major contributor to coiled-coil stability. Our results show clearly that protein stability is more complex than previously thought and unique sites can synergistically control protein stability over long distances.
Hodges
Robert S
RS
Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, 80045, USA. robert.hodges@ucdenver.edu
Mills
Janine
J
McReynolds
Susanna
S
Kirwan
J Paul
JP
Tripet
Brian
B
Osguthorpe
David
D
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D030562:Databases, Protein; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D055672:Static Electricity; D013816:Thermodynamics; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D014335:Tropomyosin