Kremneva Elena, Nikolaeva Olga, Maytum Robin, Arutyunyan Alexander M, Kleimenov Sergei Yu, Geeves Michael A, Levitsky Dmitrii I
A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia.
FEBS J. 2006 Feb;273(3):588-600. 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 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.
我们使用差示扫描量热法（DSC）和圆二色性（CD）来研究重组α-原肌球蛋白（Tm）的成纤维细胞亚型与平滑肌Tm的热解折叠情况。这两种非肌肉Tm亚型5a和5b在内部仅外显子6b/6a不同，并且它们与平滑肌Tm的区别均在于N端外显子1b取代了肌肉特异性外显子1a和2a。我们发现，外显子1b的存在显著降低了DSC观察到的Tm热解折叠的可测量热焓，尽管它对Tm的α-螺旋含量或Tm二聚体之间的端对端相互作用没有影响。结果表明，成纤维细胞Tm（而非平滑肌Tm）分子的很大一部分以非协同方式解折叠，在测量的协同热转变中热焓不再可见。另一方面，DSC和CD研究均表明，用非肌肉外显子1b取代肌肉外显子1a和2a不仅增加了Tm N端部分的热稳定性，还通过将Tm的主要热转变温度移至更高温度而显著稳定了Tm。用外显子6a取代外显子6b导致α-Tm热稳定性进一步增加。因此，我们的数据首次显示了肌肉和非肌肉α-Tm亚型在热解折叠方面的显著差异，并表明可变剪接外显子的替换改变了整个Tm分子的稳定性。
