L.-Y. Lian

Determination of the solution structures of domains II and III of protein G from Streptococcus by 1H nuclear magnetic resonance

We have used 1H nuclear magnetic resonance spectroscopy to determine the solution structures of two small (61 and 64 residue) immunoglobulin G (IgG)-binding domains from protein G, a cell-surface protein from Streptococcus strain G148. The two domains differ in sequence by four amino acid substitutions, and differ in their affinity for some subclasses of IgG. The structure of domain II was determined using a total of 478 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints; that of domain III was determined using a total of 445 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints. A protocol which involved distance geometry, simulated annealing and restrained molecular dynamics was used to determine ensembles of 40 structures consistent with these restraints. The structures are found to consist of an alpha-helix packed against a four-stranded antiparallel-parallel-antiparallel beta-sheet. The structures of the two domains are compared to each other and to the reported structure of a similar domain from a protein G from a different strain of Streptococcus. We conclude that the difference in affinity of domains II and III for IgG is due to local changes in amino acid side-chains, rather than a more extensive change in conformation, suggesting that one or more of the residues which differ between them are directly involved in interaction with IgG.

Protein-ligand interactions: Exchange processes and determination of ligand conformation and protein-ligand contacts

Publisher Summary This chapter describes exchange processes and determination of ligand conformation and protein-ligand contacts. Nuclear magnetic resonance (NMR) spectroscopy can provide information on many different aspects of protein-ligand interactions, ranging from the determination of the complete structure of a protein-ligand complex to focusing on selected features of the interactions between the ligand and protein by using reporter groups on the ligand or the protein. In addition to the structural information, dynamic, kinetic, and thermodynamic aspects of ligand binding are presented. Early analysis of ligand binding focused on measurements of relaxation times, chemical shifts, and coupling constants, which gave relatively limited, though valuable, structural information. The first step in any study of protein-ligand interactions by NMR is to establish to which region of exchange the spectrum corresponds (or, more correctly, the resonances of interest, because different resonances can show different exchange behavior).

1H, 15N and 13C NMR resonance assignment, secondary structure and global fold of the FMN-binding domain of human cytochrome P450 reductase.

The FMN-binding domain of human NADPH-cytochrome P450 reductase,corresponding to exons 3-;7, has been expressed at high level in anactive form and labelled with 13C and 15N. Mostof the backbone and aliphatic side-chain 1H, 15Nand 13C resonances have been assigned using heteronucleardouble- and triple-resonance methods, together with a semiautomaticassignment strategy. The secondary structure as estimated from the chemicalshift index and NOE connectivities consists of six α-helices and fiveβ-strands. The global fold was deduced from the long-range NOEsunambiguously assigned in a 4D 13C-resolved HMQC-NOESY-HMQCspectrum. The fold is of the alternating α/β type, with the fiveβ-strands arranged into a parallel β-sheet. The secondarystructure and global fold are very similar to those of the bacterialflavodoxins, but the FMN-binding domain has an extra short helix in place ofa loop, and an extra helix at the N-terminus (leading to the membrane anchordomain in the intact P450 reductase). The experimental constraints werecombined with homology modelling to obtain a structure of the FMN-bindingdomain satisfying the observed NOE constraints. Chemical shift comparisonsshowed that the effects of FMN binding and of FMN reduction are largelylocalised at the binding site.

Identification of the C2-1H histidine NMR resonances in chloramphenicol acetyltransferase by a 13C-1H heteronuclear multiple quantum coherence method.

Chloramphenicol acetyltransferase (CAT) was used to assess the feasibility of study of specific proton resonances in an enzyme of overall molecular mass 75000. [ring2‐13C]Histidine was selectively incorporated into the type III chloramphenicol acetyltransferase (CATIII) using a histidine auxotroph of E. coli. Heteronuclear multiple and single quantum experiments were used to select the C2 protons in the histidyl imidazole ring. One‐ and two‐dimensional spectra revealed six signals out of a total of seven histidine residues in CATIII. pH titration, chemical modification and ligand binding were used to demonstrate that the signal from H195, the histidine at the active site, is not among those observed. Nevertheless, this work demonstrates that selective isotopic enrichment and multiple quantum coherence techniques can be used to distinguish proton resonances in a protein of high molecular mass.