Thermostability of endo-1,4-beta-xylanase II from Trichoderma reesei studied by electrospray ionization Fourier-transform ion cyclotron resonance MS,hydrogen/deuterium-exchange reactions and dynamic light scattering

Endo-1,4-beta -xylanase II (XYNII) from Trichoderma reesei is a 21 kDa enzy me that catalyses the hydrolysis of xylan, the major plant hemicellulose. I t has various applications in the paper, food and feed industries. Previous thermostability studies have revealed a significant decrease in enzymic ac tivity of the protein at elevated temperatures in citrate buffer [Tenkanen, Puls and Poutanen (1992) Enzyme Microb. Technol. 14, 566-574]. Here, therm ostability of XYNII was investigated using both conventional and nanoelectr ospray ionization Fourier-transform ion cyclotron resonance MS and hydrogen /deuterium (H/D)exchange reactions. In addition, dynamic light scattering ( DLS) was used as a comparative method to observe possible-changes in both t ertiary and quaternary structures of the protein. We observed a significant irreversible conformational change and dimerization when the protein was e xposed to heat. H/D exchange revealed two distinct monomeric protein popula tions in a narrow transition temperature region. The conformational change in both the water and buffered solutions occurred in the same temperature r egion where enzymic-activity loss had previously been observed. Approx. 10- 30% of the protein was specifically dimerized when exposed to the heat trea tment. However, adding methanol to the solution markedly lowered the transi tion temperature of conformational change as well as increased the dimeriza tion up to 90%. DLS studies in water confirmed the change in conformation o bserved by electrospray ionization MS. We propose that the conformational c hange is responsible for the loss of enzymic activity at temperatures over 50 degreesC and that the functioning of the active site-in the enzyme is un feasible in a new, more labile solution conformation.