Search

NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR-derived contacts for an integral membrane protein (T1088). For the three targets with NMR-based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of "inverse structure determination," in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238-residue integral membrane porin, was also used to assess several NMR-assisted prediction methods. Most groups involved in this exercise generated similar beta-barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis., (© 2021 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

The copK gene is localized on the pMOL30 plasmid of Cupriavidus metallidurans CH34 within the complex cop cluster of genes, for which 21 genes have been identified. The expression of the corresponding periplasmic CopK protein is strongly upregulated in the presence of copper, leading to a high periplasmic accumulation. The structure and metal-binding properties of CopK were investigated by NMR and mass spectrometry. The protein is dimeric in the apo state with a dissociation constant in the range of 10(-5) M estimated from analytical ultracentrifugation. Mass spectrometry revealed that CopK has two high-affinity Cu(I)-binding sites per monomer with different Cu(I) affinities. Binding of Cu(II) was observed but appeared to be non-specific. The solution structure of apo-CopK revealed an all-beta fold formed of two beta-sheets in perpendicular orientation with an unstructured C-terminal tail. The dimer interface is formed by the surface of the C-terminal beta-sheet. Binding of the first Cu(I)-ion induces a major structural modification involving dissociation of the dimeric apo-protein. Backbone chemical shifts determined for the 1Cu(I)-bound form confirm the conservation of the N-terminal beta-sheet, while the last strand of the C-terminal sheet appears in slow conformational exchange. We hypothesize that the partial disruption of the C-terminal beta-sheet is related to dimer dissociation. NH-exchange data acquired on the apo-protein are consistent with a lower thermodynamic stability of the C-terminal sheet. CopK contains seven methionine residues, five of which appear highly conserved. Chemical shift data suggest implication of two or three methionines (Met54, Met38, Met28) in the first Cu(I) site. Addition of a second Cu(I) ion further increases protein plasticity. Comparison of the structural and metal-binding properties of CopK with other periplasmic copper-binding proteins reveals two conserved features within these functionally related proteins: the all-beta fold and the methionine-rich Cu(I)-binding site.

Y64 has been replaced in cytochrome c553 from Desulfovibrio vulgaris Hildenborough by phenylalanine, leucine, valine, serine and alanine residues. An NMR study of structural variation induced in both oxidoreduction states of the molecule has been carried out by analysing observed chemical-shift variations. Dynamic changes were evidenced using NH exchange. We have observed that the substitution has a drastic effect on the stability of the molecule in the reduced state, although there is no effect on the reduction potential of the cytochrome. Y64-->F substitution induces particular effects on the NH exchange at the N-terminal, C-terminal and central alpha-helices and increases the stability of the oxidized molecule.

The solution structure of the Ectothiorhodospira halophila ferrocytochrome c551 has been determined. This molecule belongs to a separate class of small bacterial cytochromes c for which no 3D structure has been reported so far. It is characterized by a very low redox potential (58 mV) and is isolated from the periplasm of halophilic purple phototrophic bacteria. For the 78 residue protein, 1445 NOE derived distance constraints were used in a combined simulated annealing/restrained molecular dynamics calculation. The final ensemble of 37 structures presents a backbone r.m.s.d. of less than 0.5 A compared to the mean structure. The physical viability of these structures was investigated by subjecting eight of them to a constraint free molecular dynamics simulation. No systematic conformational change was observed and the average backbone r.m.s.d. compared to the initial structures was less than 1.5 A. The structure of the E. halophila cytochrome c551 shows a striking resemblance to Azotobacter vinelandii cytochrome c5. Significant differences in backbone conformations occur in three small regions which are implicated in solvent protection of the heme propionates and thiomethyl-8(1). Comparison with Pseudomonas aeruginosa cytochrome c551 reveals that only the common cytochrome c core, i.e. three helices, is conserved. The folding of the protein chain around the heme propionates is very different and results in more efficient solvent protection in Ps. aeruginosa. The electrostatic surface of E. halophila cytochrome c551 was found to be significantly different from mitochondrial cytochromes c and bacterial cytochromes c2 but similar to that of Ps. aeruginosa cytochrome c551.

In order to study the conformational stability induced by the replacement of Tyr-64 in Desulfovibrio vulgaris Hildenborough (DvH) cytochrome c553, fast peptic digestion of deuterated protein followed by separation and measurement of related peptides using liquid chromatography coupled to electrospray ionization mass spectrometry was performed. We show that the H-bonding and/or solvent accessibility properties were modified by the single-site mutation. The mutant proteins can be classified into two groups: the Y64F and Y64L mutants with nearly unchanged deuterium incorporation compared to the wild-type protein and the Y64S, Y64V and Y64A mutants with increased deuterium incorporation. The 70-74 peptide was the most affected by mutation of Tyr-64, the phenylalanine mutant inducing slight stabilization whereas the serine mutant was significantly destabilized. In addition, from the analysis of the overlapping 37-57 and 38-57 peptides we can conclude that the amide proton of Tyr-38 has been replaced by deuterium in all proteins.

Hydrogen/deuterium exchange as well as charge state distribution monitored by electrospray ionization mass spectrometry were demonstrated to be a powerful and effective new tool for probing conformational properties of proteins in solution. In this paper, the influence of single amino acid replacements on the global conformation of cytochrome C553 from Desulfovibrio vulgaris Hildenborough using isotopic exchange monitored by electrospray ionization mass spectrometry is reported. Based on their respective charge state distributions and isotopic exchanges, we have differentiated relative stability of mutants and a ladder classification with the order being wild-type > Y64F = Y64L > Y64V > Y64A, under specific conditions of pH, is proposed.