Studies on Protein Structures using X-ray crystallography and Small-Angle X-ray Scattering

Abstract

Proteins produced from genomic information are polymeric molecules that consist of 20 amino acids in many different combinations and sequences, and they exist in enormous variety, ranging in size from small peptides to multimeric polymers. The 20 amino acids are classified by the R group located off the α carbon. The R group gives each amino acid distinct properties, from polar and hydrophilic to nonpolar and hydrophobic. Amino acids can polymerize into very long polypeptides through the formation of peptide bonds. Such polypeptides possess structures and functions depending on the particular frequency and combination of the amino acid residues, which is determined by the genetic code. In other words, the sequence of amino acids in a protein is a key that assigns the distinct structure and characteristics of each protein. Therefore, the determination of the structure of a protein can provide important information about how it functions. The most widely known methods of structural study of proteins are X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, cryo electron microscopy (EM), and small-angle X-ray scattering (SAXS). In this thesis, a structural study of protein using X-ray crystallography and SAXS methods is described.E. coli PmrD is signaling protein that can act as a specific connecter between PmrA/PmrB and PhoP/PhoQ and is a post-translational activator. The X-ray crystal structure of E. coli PmrD was determined at 2.35 Å resolution, and the structure was composed of a C-terminal α-helix and an anti-parallel β-barrel containing six β-strands. The crystal structure of PmrD revealed a disulfide between Cys9 in strand β1 and Cys81 in the α-helix. The ab initio model of E. coli PmrD was obtained using SAXS measurements. The overall structure has a rhomboid shape. The superimposition of the crystal structure and SAXS models shows that the PmrD dimer structure fits nicely with the ab initio SAXS model, and PmrD may exist as a dimer in buffer solution. The structural studies suggest that the biological assembly of E. coli PmrD is a physiological dimer, unlike the monomeric crystal structure. All electropositive surfaces of E. coli PmrD do not interact with N-terminal regulatory region of E. coli PmrA, suggesting that the PmrA/PmrB system for polymyxin B resistance only exists in E. coli. These above results indicate that the PhoP/PhoQ system in E. coli could not activate due to the dimeric formation of PmrD unlike Salmonella PmrD.Small MutS-related domains (SMRDs), which are widely conserved in all organisms, possess a nicking endonuclease activity, incising the phosphate at the 3′ backbone position of DNA. The X-ray crystal structure of the SMRD of the human BCL-3 binding protein (HSMRD) was solved at 1.9 Å resolution and assayed for nicking endonuclease activity. The HSMRD structure reveals a similar domain assembly to the catalytic domains of DNase I and RNase H1. The N-terminal extended SMRD (exHSMRD) model obtained with the SAXS technique shows an elongated shape, which is different from the globular HSMRD structure. The exHSMRD and its mutant exhibit random DNA endonucleolytic activity unlike the nicking endonuclease activity of HSMRD and its mutant form. The structural and functional differences between the nicking endonuclease HSMRD and the DNaseI-like endonuclease exHSMRD provide information on the DNA binding and nucleation of these proteins. The creatine kinase (CK) isoenzymes are members of the phosphagen kinase family and are responsible for cellular metabolism and energy homeostasis. CK isoenzymes catalyze the reversible transfer of a phosphoryl group

there are two cytosolic isoenzymes. The CK enzymes of the brain (B) and muscle (M) types can form homodimers (CK-BB and CK-MM) or heterodimers (CK-MB). Structural studies using SAXS technique were performed for monomeric CK-M, CK-B, and heterodimeric CK-MB. The ab initio SAXS models of CK-M (Rg,p(r): 33.01 Å) and CK-B (Rg,p(r): 33.28 Å) look like bent right and left hands, respectively. The superimposition results show that the ab initio models have structural similarities to the crystal structures. Additionally, the ab initio model of human CK-MB heterodimer was first determined, and the structural information for CK-MB was reported. The overall structure of CK-MB (Rg,p(r): 50.75 Å) looks like the bent hands have connected their fingers (N-terminal regions of CK-B and CK-M). SAXS analysis of CK-MB indicated that the dimerization of CK-MB required N-terminal conformational changes compared to the crystal structure of the monomeric state.