Tropomyosin (Tpm) is an actin-binding protein that plays a vital role in the regulation of muscle contraction. Fast skeletal muscles express 2 Tpm isoforms, a (Tpm 1.1) and b (Tpm 2.2), resulting in the existence of 2 forms of dimeric Tpm molecule: Aa-homodimer and αβ-heterodimer. ββ-Homodimer is unstable and absent in the native state,despitewhichmost of the studies ofmyopathy-relatingTpmmutations have been performed on the ββ-homodimer. Here, we applied different methods to investigate the effects of myopathic mutations R133Wand N202K in the β-chain of Tpm on properties of αβ-heterodimers and to compare them with the features of ββ-homodimers with the same mutations. The results show that properties of αβ-Tpm and ββ-Tpm with substitutions in the β-chain differ significantly, and this indicates that the effects of myopathic mutations in the Tpm β-chain should be studied on the Tpm αβ-heterodimer.