A Study on the Temperature-Dependent Behavior of Small Heat Shock Proteins from Methanogens

Abstract

Small heat shock proteins (sHsps) are ubiquitous low-molecular-weight chaperones that prevent protein aggregation under cellular stress conditions. In the absence of stress, they assemble into large oligomers. In response to stress, such as elevated temperatures, they undergo conformational changes that expose hydrophobic surfaces, allowing them to interact with denatured proteins. At heat shock temperatures in bacteria, large sHsp oligomers disassemble into smaller oligomeric forms. Methanogens are a diverse group of microorganisms, ranging from thermophilic to psychrophilic and halophilic species. Accordingly, their sHsps exhibit markedly different temperature dependencies based on their optimal growth temperatures. In this study, we characterized sHsps from both hyperthermophilic and mesophilic methanogens to investigate the mechanisms underlying their temperature-dependent behavior. Using analytical ultracentrifugation, we observed the dissociation of sHsps from a mesophilic methanogen into dimers. The dissociation equilibrium of these oligomers was found to be dependent not only on temperature but also on protein concentration. Furthermore, by generating various mutants, we identified the specific amino acid residues responsible for the temperature dependency observed. The C-terminal region containing the IXI/V motif and the α-crystallin domain were found to be the primary determinants of oligomer stability and its temperature dependence.

Keywords: analytical ultracentrifuge; chaperone; methanogen; small heat shock protein; stress.

Figure 1

Sequence alignment and HPLC-SEC of MMsHsp, MJsHsp, NMCJsHsp, and NJCMsHsp. (a) Sequence alignment of MMsHsp, MJsHsp, NMCJsHsp, and NJCMsHsp. The T33M mutation of MJsHsp is demonstrated in blue. The N-terminal region is enclosed in a green box. The amino acids that were mutated in this study are marked in red. “*” indicates strictly conserved residues, “:” indicates strongly similar residues, and “.” indicates weakly similar residues. (b) Oligomeric structures of MMsHsp, MJsHsp, NMCJsHsp, and NJCMsHsp were analyzed by HPLC-SEC at various temperatures. A total of 50 µL of 40 µM sHsp was applied to SB-804HQ, and absorbance was monitored at 280 nm at 20 °C and at elevated temperatures (40 °C, 50 °C, and 60 °C). A.U.: arbitrary unit.

Figure 2

AUC results of MJsHsp (a) and MMsHsp (b) at 40 °C. Blue and red lines represent c(s_20,w), indicating the results at 1.0 mg/mL and 0.1 mg/mL, respectively. Peaks a–h correspond to those listed in Table 1.

Figure 3

Chaperone function of MMsHsp and NJCMsHsp. The thermal aggregation of CS from a porcine heart was monitored by measuring light scattering at 500 nm with a spectrofluorometer at 50 °C. CS (50 nM, monomer) was incubated in an assay buffer with or without MMsHsp (a) or NJCMsHsp (b) at the specified concentration (600 nM and 1200 nM). (c) A total of 50 µM of insulin was treated with DDT, with or without MMsHsp or NJCMsHsp at the specified molar ratio. Aggregation of insulin was monitored by measuring light scattering at 360 nm with a spectrofluorometer at 30 °C.

Figure 4

HPLC-SEC of single mutants of MMsHsp and MIsHsp. Oligomeric structures of single mutants of MMsHsp and MJsHsp were analyzed via HPLC-SEC at various temperatures. A total of 50 µL of the 40 µM sHsp variant was applied to SB-804HQ, and absorbance was monitored at 280 nm at 20 °C and at elevated temperatures (40, 50, and 60 °C).

Figure 5

HPLC-SEC of the double, triple, and quadruple mutants, MMsHsp-Chimera and MJsHsp-Cmut. Oligomeric structures of single mutants of MMsHsp and MJsHsp were analyzed via HPLC-SEC at various temperatures. A total of 50 µL of the 40 µM sHsp variant was applied to SB-804HQ, and absorbance was monitored at 280 nm at 20 °C and at elevated temperatures (40, 50, and 60 °C).

Figure 6

Sequence alignment and HPLC-SEC of NMCJsHsp-Mut. (a) Sequence alignment of NMCJsHsp-Mut and NJsHsp. T33M mutation of MJsHsp is shown in blue. The mutated amino acids of MCJsHsp-Mut are marked in red. “*” indicates strictly conserved residues, “:” indicates strongly similar residues, and “.” indicates weakly similar residues. The N-terminal region is indicated by a black box. (b) Oligomeric structures of NMCJsHsp-Mut were analyzed via HPLC-SEC. A total of 50 µL of 40 µM of NMCJsHsp-Mut was applied to SB-804HQ, and absorbance was monitored at 280 nm at 20 °C and at elevated temperatures (40 °C, 50 °C, and 60 °C).