19F NMR spectroscopy monitors ligand binding to recombinantly fluorine-labelled b'x from human protein disulphide isomerase (hPDI)

We report a protein-observe (19)F NMR-based ligand titration binding study of human PDI b'x with ?-somatostatin that also emphasises the need to optimise recombinant protein fluorination when using 5- or 6-fluoroindole. This study highlights a recombinant preference for 5-fluoroindole over 6-fluoroindole; most likely due to the influence of fluorine atomic packing within the folded protein structure. Fluorination affords a single (19)F resonance probe to follow displacement of the protein x-linker as ligand is titrated and provides a dissociation constant of 23 ± 4 ?M

Biophysical characterization and NMR analysis of the PDI fragment b'xa'c

Protein disulphide isomerase (PDI) was the first catalyst of protein folding to be identified and is a key cellular chaperone required for the rapid formation of correctly folded disulphide bonded proteins in the secretory pathway. PDI is the archetype of a large family of ER-resident PDI-like proteins and contains four thioredoxin-like domains, two of which, like thioredoxin itself, have redox-active (COHC) catalytic sites (a and a') and two of which do not (b and b'). The domain order is abb'xa'c where x is a 19 residue linker between the b' and a' domains and c is a C-terminal acidic tail containing the KDEL ER retention signal. The x linker has been shown to occlude the primary ligand binding site in the human b'x construct; an event called "capping". In previous work, site-directed mutants were identified that stabilise both the capped and uncapped conformations of b'x. Furthermore, the binding of x was shown to compete with peptide ligands and could prove to be an important physiological mechanism controlling access to the hydrophobic binding site. The movement of the x linker in gating the binding site could also lead to wider structural arrangement of the protein and control other aspects of PDI structure and function. This study focuses on the characterisation of the smallest PDI fragment with isomerase activity, b'xa'c, to further understand the role of x capping and its int~raction with adjacent b' and a' domains. Mutants ofb'xa'c, which in b'x favoured capping or uncapping of the ligand binding site, were generated by site-directed mutagenesis. All b'xa'c proteins presented as a mixture of monomer and dimer, which on gel filtration presented with larger hydrodynamic volumes than expected for globular proteins of similar sizes. Denaturation studies using guanidine hydrochloride showed that the capping 1272A mutant of b'xa'c presented with a higher conformational stability than WT b'xa'c, whereas the uncapping mutant L343A was less conformationally stable than WT and 1272A b'xa'c. b'xa'c proteins presented with a biphasic denaturation curve in which the first phase could be attributed to the unfolding of the a' domain and the second phase was due to b'x unfolding. NMR studies of b'xa'c proved challenging due to broad and poorly resolved spectra. Studies on b'x and x'ac were carried out as a stepping stone to the investigations of b'xa'c. The oxidation state of the a' domain, temperature and pH had a significant effect on the line widths seen for b'xa'c, and allowed the backbone resonance assigrunent ofxa'c and I272A b'xa'c to be carried out. Relaxation dynamics studies showed that the a' domain has a more flexible backbone than b'x and that neighbouring domains affect the conformational behaviour of one another. NMR and intrinsic fluorescence showed no conclusive evidence of capping of the ligand binding site by the x linker region in b'xa'c, but mutants 1272A, L343A and D346A1D34A had a significant effect on the conformational stability of b'xa'c.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Biophysical characterization and NMR analysis of the PDI fragment b'xa'c

Protein disulphide isomerase (PDI) was the first catalyst of protein folding to be identified and is a key cellular chaperone required for the rapid formation of correctly folded disulphide bonded proteins in the secretory pathway. PDI is the archetype of a large family of ER-resident PDI-like proteins and contains four thioredoxin-like domains, two of which, like thioredoxin itself, have redox-active (COHC) catalytic sites (a and a') and two of which do not (b and b'). The domain order is abb'xa'c where x is a 19 residue linker between the b' and a' domains and c is a C-terminal acidic tail containing the KDEL ER retention signal. The x linker has been shown to occlude the primary ligand binding site in the human b'x construct; an event called "capping". In previous work, site-directed mutants were identified that stabilise both the capped and uncapped conformations of b'x. Furthermore, the binding of x was shown to compete with peptide ligands and could prove to be an important physiological mechanism controlling access to the hydrophobic binding site. The movement of the x linker in gating the binding site could also lead to wider structural arrangement of the protein and control other aspects of PDI structure and function. This study focuses on the characterisation of the smallest PDI fragment with isomerase activity, b'xa'c, to further understand the role of x capping and its interaction with adjacent b' and a' domains. Mutants of b'xa'c, which in b'x favoured capping or uncapping of the ligand binding site, were generated by site-directed mutagenesis. All b'xa'c proteins presented as a mixture of monomer and dimer, which on gel filtration presented with larger hydrodynamic volumes than expected for globular proteins of similar sizes. Denaturation studies using guanidine hydrochloride showed that the capping 1272A mutant of b'xa'c presented with a higher conformational stability than WT b'xa'c, whereas the uncapping mutant L343A was less conformationally stable than WT and 1272A b'xa'c. b'xa'c proteins presented with a biphasic denaturation curve in which the first phase could be attributed to the unfolding of the a' domain and the second phase was due to b'x unfolding. NMR studies of b'xa'c proved challenging due to broad and poorly resolved spectra. Studies on b'x and x'ac were carried out as a stepping stone to the investigations of b'xa'c. The oxidation state of the a' domain, temperature and pH had a significant effect on the line widths seen for b'xa'c, and allowed the backbone resonance assignment of xa'c and I272A b'xa'c to be carried out. Relaxation dynamics studies showed that the a' domain has a more flexible backbone than b'x and that neighbouring domains affect the conformational behaviour of one another. NMR and intrinsic fluorescence showed no conclusive evidence of capping of the ligand binding site by the x linker region in b'xa'c, but mutants 1272A, L343A and D346A1D34A had a significant effect on the conformational stability of b'xa'c

Biophysical characterization and NMR analysis of the PDI fragment b'xa'c

Protein disulphide isomerase (PDI) was the first catalyst of protein folding to be identified and is a key cellular chaperone required for the rapid formation of correctly folded disulphide bonded proteins in the secretory pathway. PDI is the archetype of a large family of ER-resident PDI-like proteins and contains four thioredoxin-like domains, two of which, like thioredoxin itself, have redox-active (COHC) catalytic sites (a and a') and two of which do not (b and b'). The domain order is abb'xa'c where x is a 19 residue linker between the b' and a' domains and c is a C-terminal acidic tail containing the KDEL ER retention signal. The x linker has been shown to occlude the primary ligand binding site in the human b'x construct; an event called "capping". In previous work, site-directed mutants were identified that stabilise both the capped and uncapped conformations of b'x. Furthermore, the binding of x was shown to compete with peptide ligands and could prove to be an important physiological mechanism controlling access to the hydrophobic binding site. The movement of the x linker in gating the binding site could also lead to wider structural arrangement of the protein and control other aspects of PDI structure and function. This study focuses on the characterisation of the smallest PDI fragment with isomerase activity, b'xa'c, to further understand the role of x capping and its int~raction with adjacent b' and a' domains. Mutants ofb'xa'c, which in b'x favoured capping or uncapping of the ligand binding site, were generated by site-directed mutagenesis. All b'xa'c proteins presented as a mixture of monomer and dimer, which on gel filtration presented with larger hydrodynamic volumes than expected for globular proteins of similar sizes. Denaturation studies using guanidine hydrochloride showed that the capping 1272A mutant of b'xa'c presented with a higher conformational stability than WT b'xa'c, whereas the uncapping mutant L343A was less conformationally stable than WT and 1272A b'xa'c. b'xa'c proteins presented with a biphasic denaturation curve in which the first phase could be attributed to the unfolding of the a' domain and the second phase was due to b'x unfolding. NMR studies of b'xa'c proved challenging due to broad and poorly resolved spectra. Studies on b'x and x'ac were carried out as a stepping stone to the investigations of b'xa'c. The oxidation state of the a' domain, temperature and pH had a significant effect on the line widths seen for b'xa'c, and allowed the backbone resonance assigrunent ofxa'c and I272A b'xa'c to be carried out. Relaxation dynamics studies showed that the a' domain has a more flexible backbone than b'x and that neighbouring domains affect the conformational behaviour of one another. NMR and intrinsic fluorescence showed no conclusive evidence of capping of the ligand binding site by the x linker region in b'xa'c, but mutants 1272A, L343A and D346A1D34A had a significant effect on the conformational stability of b'xa'c.EThOS - Electronic Theses Online ServiceGBUnited Kingdo