ANALYSIS OF HUMAN RED-CELL SPECTRIN TETRAMER (HEAD-TO-HEAD) ASSEMBLY USING COMPLEMENTARY UNIVALENT PEPTIDES

The mass-driven assembly of spectrin dimers to form tetramers involves two equal head-to-head alpha-beta associations and requires at least 30-degrees-C for interconversion to occur readily. In this paper, the properties of tetramer formation were investigated using two complementary univalent peptides (the alphaI domain and beta monomers). Since the al domain lacks an essential nucleation site required for side-to-side (lateral) heterodimer assembly [Speicher et al. (1992) J. Biol. Chem. 267, 14775-14782], these two peptides can only assemble head-to-head at a single site. This head-to-head assembly readily occurs at lower temperatures, indicating the temperature barrier for dimer-tetramer interconversion is caused by a conformational constraint of the dimer. This constraint, a closed hairpin loop, is released when the laterally associated partner is removed. The univalent alphaI-beta binding affinity at 37-degrees-C (K(a) = 1.4 x 10(5) M-1) is similar to the dimer-tetramer association constant at the same temperature. As the temperature is decreased from 37 to 0-degrees-C, the alphaI-beta binding affinity increases about 32-fold. In contrast with head-to-head associations involving dimers, the second-order rate constants of two complementary univalent peptides (i.e., alphaI and beta) are dramatically higher, and the estimated activation energy (about 50 kJ mol-1) is about 5-fold lower. An open dimer conformation is an obligatory high-energy intermediate required for dimer-tetramer interconversion, and opening the dimer hairpin loop contributes about 190 kJ mol-1 to the activation energy for tetramer association. Since closed dimers probably contain an internal head-to-head alpha-beta association, the closed reversible open dimer equilibrium would shift toward closed dimers at lower temperatures and would dramatically slow dimer-tetramer interconversion at low temperatures. The current model defines the relative contributions of each alpha-beta association in dimer-tetramer interconversion, and it also allows estimations of the relative proportions of open and closed dimers under different experimental and physiological conditions.