Structural Studies of β-microseminoproteins and of Lon Protease α-domain in Complex with DNA

• Main Authors: Iren Wang, 王怡人
• Other Authors: 吳世雄
• Format: Others
• Language: en_US
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/32297389746944694247

博士 === 國立臺灣大學 === 生化科學研究所 === 95 === It is well known that NMR, in addition to X-ray crystallography, is another powerful technique used to determine the 3D structure of biomacromolecule. In this dissertation, several recombinant unlabeled as well as 15N- or 15N/13C-labeled proteins were expressed and purified in large quantities. We then carried out circular dichroism (CD) and heteronuclear multidimensional NMR experiments to gain insight into the structure-function relationships on these proteins, as described below. Part I. β-microseminoproteins (MSPs), identified from diverse species, are all non-glycosylated and disulfide bond-rich, but show a relatively low level of conservation. Although all Cys residues are conserved, the disulfide bond pairings of porcine MSP determined based on NOEs are different from those of ostrich MSP derived based on mass spectrometric analysis. CD titration spectra revealed that both porcine and human MSPs are thermally and chemically stable. The solution structure of porcine MSP determined on the basis of 1018 restraints showed that it exhibits a β-sheet-rich structure with two distinct domains, an N-terminal domain consisting of one double-stranded and one four-stranded antiparallel β-sheets, and a C-terminal domain consisting of two double-stranded antiparallel β-sheets. The orientation of the two domains was derived mainly on the basis of long-range NOEs and verified using residual dipolar coupling data. A number of charged residues were found in close proximity between the two domains, indicating that electrostatic interaction may be the key contact between the two domains. Also, structure of porcine MSP is the first 3D structure reported among all MSPs, and contains a novel fold according to the structural comparison using DALI, CATH, and CE methods. Part II. Lon protease is mainly responsible for eliminating misfolded or damaged proteins in cells. A small α-domain, localized in the sub-domain of the ATPase domain for Lon, is thought to carry the substrate-recognition and DNA-binding sites. CD spectra revealed that secondary structure and structural stability of Lon α-domains are dramatically altered at extreme pH values. Solution structure of the Bacillus subtilis Lon α-domain at pH 5.8 is consisted of four α-helices and a short two-stranded parallel β-sheet. Although this α-domain contains neither the canonical helix-turn-helix motif nor the winged-helix motif, as usually observed in the DNA-binding protein, it was confirmed that this α-domain indeed binds with DNA based on gel mobility shift assay and NMR shift perturbation experiment. It is suggested based on NMR data that the positively charged residues in α3 helix and in the both loops connecting with α3 are the major sites responsible for DNA binding. Structural comparisons of various Lon α-domains further revealed that other Lon α-domains are very likely to possess similar DNA-binding characteristics.