The Stucture Of Starch Binding AmyP-SBD And The Salt Adaption Mechanism Of The Ubiquitin Like Protein SAMP2

Starch binding domains （SBDs） are important for the functions of alpha -amylases. AmyP which is a novel alpha-amylase was isolated from a marine metagenomic library. AmyP was shown to exhibit a unique ability to rapidly digest raw rice starch, which is first described in known alpha-amylases. The discovery led to the identification of a new subfamily alpha-amylase family （GH13₃7） that may be an independent clade of ancestral marine bacterial alpha-amylases. Preliminary sequence analysis and function study indicated that at the C-terminus of AmyP is an SBD. Low similarity of the SBD sequence with that of any classified CBM indicates that this SBD defines a novel family of CBMs, which was named CBM69. The SBD has an unexpectedly stronger effect on the catalytic activity toward soluble starch than raw starch, indicating that the SBD plays an important role in soluble starch hydrolysis, which broadens our understanding of the function of SBDs.Our previous isothermal titration calorimetry （ITC） experiment showed that there was a strong interaction between AmyP-SBD and substrate analogue β-cyclodextrin （β-CD）, and nuclear magnetic resonance （NMR） result showed that free form AmyP-SBD adopted a flexible state composed of interconverting well folded conformations, which converged to form a unique tertiary structure when binding to β-cyclodextrin. This substrate induced folding isvery rare in other SBDs. Study on the structure of AmyP-SBD will be helpful to explain the mechanism of AmyP degradation and its substrate induced folding. In the work of this thesis, we analyzed the three-dimensional structure of AmyP-SBD complexed with β-cyclodextrin by NMR method. The chemical shifts of the main chainatoms have been assigned, and the preliminary calculation results show that the structure is similar to other SBDs, but there is a significant difference in topology and other terms, which may be related to its unique characteristics. Fine structure calculation is ongoing.It is of great significance to explain the mechanisms of formation of biodiversity and biological adaptation to extreme environments, both in theory and practice. Elucidation of halophilic mechanism of proteins will contribute to it. In this regard, there are a few main viewpoints, concerning surface charge, hydrophobicity, and side chain length of the residues respecitively. However, are these mechanisms applicable to all halophilic proteins? Is there any other mechanism? All these quetions need to be addressed. SAMP2 protein, isolated from a halophilic archear Haloferax volcanii, is identified as a ubiquitin-like protein. Its solution and crystal structures under high salt conditions have been solved. The results show that it is similar to the eukaryotic ubiquitin-like proteins, adopting aconserved beta-grasp fold. Notably, under low salt conditions, SAMP2 protein adopts two main conformations, one is ordered, same as that under high salt conditions, and the other is completely disordered, similar to the bacterial Ubiquitin-like protein Pup. With the salt concentration increased, the equilibrium of the two conformations transferred gradually to the ordered one. In this thesis, we used site directed mutagenesis and directed evolution method to investigate the effect of matation on SAMP2 structure and its salt dependence, testing the applicability of the above mechanisms and revealing other potential halophilic mechanism. Hopefuly, the study was expected to provide clues to elucidate the evolution of ubiquitin like proteins’structure and function. The results showed that increasing side chain length and hydrophobicity according to the known mechanisms did not make the SAMP2 mutants more adapted to low salt environments, but rather a protein folded better in low salt environments screened from directed evolution seemed to negate the mechanisms. More in-depth studies are needed to clarify the problem.