Spectral Studies On The Effect Of Heme On The Conformation Of Myoglobin

Myoglobin(Mb) is the protein which is studied thoroughly on the structure and function. Two components constitute Mb, globin and the prosthetic group (heme).Globin has eightα-helix,small amount ofβ-turn and random coil. The function of Mb and the structural interaction between globin and heme have been reported clearly.Amyloid fibril is a one-dimentional crystal-like proteinaceous aggregation, which is rich inβ-sheet. In 2002, Fandrich et al.found that apoMb is able to form amyloid fibril easily in pH9 solution including 50mM sodium borate at 55℃-75℃.We studied on the contribution of heme to the stability of globin confomation and got the time-course spectrum of both Mb and apoMb during the first 120min at 25,65,85℃by ATR-FTIR, CD and ThT fluorescence.The results show that at 25℃,both Mb and apoMb don't form amyloid fibril. At 65℃the globin of Mb doesn't form amyloid fibril while apoMb forms fast. This point elucidates that the existence of heme inhibits unfolding of globin and forming of amyloid fibril.At 85℃, the heme cannot prevent unfolding, aggregation and forming of amyloid fibril completely.In addition, we give the hypothesis of the forming steps of amyloid fibril. At first, the globin unfolds, exposes theα-lelix in the AGH subdomain,which is the compacted core of the globin.It's coherent with the faster reducing atθ208 thanθ222 in CD spectrum.Then the protein molecules aggregate to form protofilaments,in which the proteins remain the native secondary structure expecept the interaction parts.The interaction parts becomeβ-sheets,which are detected by ATR-IR when protofilaments aggregate to the surface of ATR-crystal .The ThT spectrum also shows the fast increasing of aggregation .This step is within 45min in both ThT and ATR-IR spectrum in our experiments. After 45min, when the protofilaments twist to mature fibrils, some stuctures of protein change fromα-helix toβ-sheet which is inside the fibril ,not binded by ThT to give the emission at 485nm.So in this step,the spectrum of ATR-IR and ThT change slowly. Our research elucidates that heme has the temperature-course contribution to the stability of globin.Heme is able to interact with globin by some forces (coordinate bonds, ion bonds, hydrophobic interactions,π-πinteractions) to stabilize the native structure of globin , hinder the domain swapping and eventually inhibit the forming of amyloid fibril which is induced by partial unfolding.