Thermal unfolding of smooth muscle and nonmuscle tropomyosin alpha-homodimers with alternatively spliced exons.
We used differential scanning calorimetry (DSC) and circular dichroism (CD) to investigate thermal unfolding of recombinant fibroblast isoforms of alpha-tropomyosin (Tm) in comparison with that of smooth muscle Tm. These two nonmuscle Tm isoforms 5a and 5b differ internally only by exons 6b/6a, and they both differ from smooth muscle Tm by the N-terminal exon 1b which replaces the muscle-specific exons 1a and 2a. We show that the presence of exon 1b dramatically decreases the measurable calorimetric enthalpy of the thermal unfolding of Tm observed with DSC, although it has no influence on the alpha-helix content of Tm or on the end-to-end interaction between Tm dimers. The results suggest that a significant part of the molecule of fibroblast Tm (but not smooth muscle Tm) unfolds noncooperatively, with the enthalpy no longer visible in the cooperative thermal transitions measured. On the other hand, both DSC and CD studies show that replacement of muscle exons 1a and 2a by nonmuscle exon 1b not only increases the thermal stability of the N-terminal part of Tm, but also significantly stabilizes Tm by shifting the major thermal transition of Tm to higher temperature. Replacement of exon 6b by exon 6a leads to additional increase in the alpha-Tm thermal stability. Thus, our data show for the first time a significant difference in the thermal unfolding between muscle and nonmuscle alpha-Tm isoforms, and indicate that replacement of alternatively spliced exons alters the stability of the entire Tm molecule.
Kremneva
Elena
E
A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia.
Nikolaeva
Olga
O
Maytum
Robin
R
Arutyunyan
Alexander M
AM
Kleimenov
Sergei Yu
SY
Geeves
Michael A
MA
Levitsky
Dmitrii I
DI
D000199:Actins / Q000502:physiology; D017398:Alternative Splicing; D000818:Animals; D002152:Calorimetry, Differential Scanning / Q000379:methods; D002942:Circular Dichroism / Q000379:methods; D003001:Cloning, Molecular; D019281:Dimerization; D005091:Exons / Q000235:genetics*; D005347:Fibroblasts / Q000502:physiology; D018398:Homeodomain Proteins / Q000235:genetics* / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014335:Tropomyosin / Q000235:genetics* / Q000502:physiology*
D000199:Actins; D018398:Homeodomain Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C490773:Tpm1 protein, rat; D014335:Tropomyosin