The residues associated with substrate binding in GroupⅠ chaperonin GroEL are hydrophobic. However, the corresponding residues in groupⅡ chaperonin ATcpnβ from Acidianus tengchongensis are hydrophilic.When these hydrophilic residues in ATcpnβ were mutated to hydrophobic residues, i.e. residues 236 from G to Y, 237 from M to F, 288 from D to L, 295 from A to V, 313 from A to V and 314 from K to V, they enhanced the inhibitory effect of ATcpnβ on the refolding of acid-denatured GFP and the inhibition activity of citrate synthase (CS) thermal aggregation. Hydrophobic interaction may contribute more to peptides binding affinity both in groupⅠ and groupⅡ chaperonin. Chaperonins have been proved to have ATP-dependent peptides refolding ability. However, it is still unclear whether peptides binding ability of chaperonins is ATP-dependent. Surface plasma resonance (SPR) analysis is used to test chaperonin binding ability to different peptides with different denaturing level. These assays revealed that ATcpnβ could capture guanidine hydrochloride denatured malate dehydrogenase in a Mg²⁺/ATP independent manner while it bind thermal aggregated citrate synthase or lysozyme in a Mg²⁺/ATP dependent manner. It has been proposed that chaperonin conformatinal changes induced by Mg²⁺/ATP to expose more hydrophobic surface is required for chaperonin capturing thermal aggregated peptides which has larger hydrophobic surfaces.