PHYSICAL-PROPERTIES OF A SINGLE-MOTIF ERYTHROCYTE SPECTRIN PEPTIDE - A HIGHLY STABLE INDEPENDENTLY FOLDING UNIT

Spectrin is a long flexible rod-like actin cross-linking protein mostl y comprised of many tandem homologous 106-residue motifs. In this stud y, the conformational stability and physical properties of a single ho mologous motif peptide, alpha 1, were evaluated and compared to intact spectrin monomers and alpha beta heterodimers. It is interesting that while spectrin dimers elongate by about 3-fold in low ionic strength buffers relative to their size in physiological buffers, the single-mo tif peptide does not show significant changes in secondary structure i n 10 mM phosphate buffer compared with isotonic buffer. This single-mo tif peptide is monomeric in physiological buffer as demonstrated by eq uilibrium sedimentation studies, and its hydrodynamic radius determine d by gel filtration and dynamic light scattering of about 2.2 nm is co nsistent with an elongated rod-like shape. Unfolding of the single-mot if peptide in urea solutions was similar to unfolding of intact hetero dimers. Differential scanning calorimetry analyses showed that this si ngle motif undergoes a reversible two-state transition with a T-m of 5 3 degrees C and an enthalpy of 65 kcal/mol in physiological buffer. Th ermal stability was unaffected by ionic strength changes, but was decr eased below physiological pH. These data show that this 13 kDa spectri n motif is a monomeric, highly stable, triple-helical, independently f olding protein building block with physical characteristics that defin e many of the structural properties of the 526 kDa spectrin heterodime r. In contrast, interactions between adjacent motifs are probably resp onsible for spectrin's molecular flexibility and elasticity.