Tài liệu Báo cáo khoa học: Thermal unfolding of smooth muscle and nonmuscle tropomyosin a-homodimers

Thermal unfolding of smooth muscle and nonmuscle

tropomyosin a-homodimers with alternatively spliced

exons

Elena Kremneva1

, Olga Nikolaeva2

, Robin Maytum3

*, Alexander M. Arutyunyan2

,

Sergei Yu. Kleimenov1

, Michael A. Geeves3 and Dmitrii I. Levitsky1,2

1 A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia

2 A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia

3 Department of Biosciences, University of Kent at Canterbury, UK

Tropomyosins (Tm) are a family of actin-binding,

a-helical coiled-coil proteins found in most eukaryotic

cells [1]. They bind to actin cooperatively along the

length of actin filaments and confer cooperativity

upon the interaction of actin with myosin heads [2].

The Tm molecules are parallel homo- or hetero￾dimers (encoded from the same or different genes) of

two a-helical chains of identical length, although the

length can vary according to isoform type. In mam￾malian cells, alternative splicing produces a variety

of muscle and nonmuscle isoforms from four differ￾ent genes [1]. Muscle cells express two major iso￾forms of Tm (a and b), each containing 284

residues. Smooth and skeletal muscles express differ￾ent isoforms resulting from alternative splicing of the

a and b genes.

There are two major classes of a-Tm: long (or high

relative molecular mass) Tm (284 residues) are

expressed in muscle and nonmuscle cells whereas short

(or low relative molecular mass) Tm (247 residues) are

Keywords

tropomyosin; actin; thermal unfolding;

differential scanning calorimetry; circular

dichroism

Correspondence

D.I. Levitsky, A.N. Bach Institute of

Biochemistry, Russian Academy of

Sciences, Leninsky prosp. 33,

119071 Moscow, Russia

Fax: +7095 9542732

E-mail: [email protected]

*Present address

School of Biological Sciences, Queen Mary,

University of London, Mile End Road,

London E1 4NS, UK

(Received 14 September 2005, revised

2 December 2005, accepted 6 December

2005)

doi:10.1111/j.1742-4658.2005.05092.x

We used differential scanning calorimetry (DSC) and circular dichroism

(CD) to investigate thermal unfolding of recombinant fibroblast isoforms

of a-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 a-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 stud￾ies 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 a-Tm thermal stability. Thus, our data show for

the first time a significant difference in the thermal unfolding between

muscle and nonmuscle a-Tm isoforms, and indicate that replacement of

alternatively spliced exons alters the stability of the entire Tm molecule.

Abbreviations

CD, circular dichroism; DSC, differential scanning calorimetry; Tm, tropomyosin; smTm, recombinant smooth muscle Tm; Tm5a and Tm5b,

recombinant fibroblast Tm isoforms with alternatively spliced exons 6b and 6a, respectively.

588 FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS