Kangcheng Ruan

High pressure static fluorescence to study macromolecular structure-function.

Through some typical examples, the high pressure static fluorescence method is described. The potentiality of the intrinsic and extrinsic fluorescence probes are analyzed for structural characterizations. Special attention is given to the use of fluorescence to understand the behavior of enzymatic reactions under high pressure. The application of fluorescence polarization is also presented together with some relevant spectroscopic problems inherent in data interpretation.

Non-specific deadenylation and deguanylation of naked RNA catalyzed by ricin under acidic condition

Ricin A-chain catalyzes the hydrolysis of the N-glycosidic bond of a conserved adenosine residue at position 4324 in the sarcin/ricin domain of 28S RNA of rat ribosome. The GAGA tetraloop closed by C-G pairs is required for recognition of the cleavage site on 28S ribosomal RNA by ricin A-chain. In this study, ricin A-chain (reduced ricin) exhibits specific depurination on a synthetic oligoribonucleotide (named SRD RNA) mimic of the sarcin/ricin domain of rat 28S ribosomal RNA under neutral and weak acidic conditions. Furthermore, the activity of intact ricin is also similar to that of ricin A-chain. However, under more acidic conditions, both enzymes lose their site specificity. The alteration in specificity of depurination is not dependent on the GAGA tetraloop of SRD RNA. A higher concentration of KCl inhibits the non-specific N-glycosidase activity much more than the specific activity of ricin A-chain. In addition, characterization of depurination sites by RNA sequencing reveals that under acidic conditions ricin A-chain can release not only adenines, but also guanines from SRD RNA or 5S ribosomal RNA. This is the first report of the non-specific deadenylation and deguanylation activity of ricin A-chain to the naked RNA under acidic conditions.

The Release of Extrinsic Polypeptides and Manganese Cluster from Photosystem 2 Membranes under High Hydrostatic Pressure

Three extrinsic polypeptides and manganese cluster were sequentially released from the membrane when photosystem 2 (PS2) membranes were kept under high hydrostatic pressure. The 17 kDa polypeptide was the most sensitive, while the 33 kDa polypeptide was the most reluctant to the treatment with high pressure. The release of manganese was not simply correlated with the loss of 33 kDa polypeptide. The losing of oxygen-evolving activity of PS2 was synchronised with the releasing of extrinsic polypeptides and manganese.

Low pH-induced conformational changes in 33 kD protein of photosystem II

Abstract33 kD protein, located on the lumen side of thylakoid membranes, is one of three extrinsic proteins of photosystem II (PS II). Previous study showed that NBS modification of W241, the only tryptophan in 33 kD protein, is helpful for understanding the function of W241 in maintaining functional conformation of 33 kD protein. In this paper, studies of both circular dichroism and fluorescence spectra showed that upon decreasing pH from 6.2 to 2.5, the conformation of soluble 33 kD protein changed significantly, with an increase or a decrease in percentage of random coil or α-helix and turns. The changes in secondary structures of this protein are pH reversible. After NBS modification at pH 2.5, the conformational change of 33 kD protein was kept fixed. The CD ellipticity at 200 nm for NBS-modified 33 kD protein is much lower than that for control, indicating that the unfolding degree of 33 kD protein was enhanced after the NBS modification. Moreover, the conformational flexibility is lost in NBS-modified 33 kD protein, and the conformational change becomes pH irreversible, indicating that NBS modification blocked the reversibility of conformational change of 33 kD protein. The specific binding capability of NBS-modified 33 kD protein is much lower than that of low pH-treated control. Furthermore, the rebinding of modified protein on PS II membranes cannot restore the activity of oxygen evolution. We suggest that it is low pH but not NBS modification of W241 that leads to the conformational change of 33 kD protein from one functional to another non-functional state. The significant capability of proton transport of 33 kD protein is discussed.