Isolation is reported of the four mutant proteins of the electron-transfer protein rubredoxin from Clostridium pasteurianum in which each of the four cysteine ligands is changed in turn to serine. They fall into two pairs whose properties depend on whether an interior (C6, C39) or a surface (C9, C42) cysteine ligand is substituted. A crystal structure of the oxidized C42S protein (1.65 Å; R, 18.5%) confirms the presence of an FeIII(Sγ-Cys)3(Oγ-Ser) center (Fe−O, 1.82(8) Å). Significant structural change is restricted to the region around the mutation. EXAFS experiments confirm FeIIIS3O (O = Oγ-Ser or OH x ) centers in each oxidized protein at pH 8. The reduction potentials of the FeIII/II couple are decreased by about 100 and 200 mV, respectively, in the interior and surface ligand mutants. The potentials are pH-dependent with respective pK a red values of about 9 and 7. EXAFS data indicate an increase of 0.2−0.3 Å in the FeII−O distances in passing through these characteristic pK a red values. 1H NMR experiments on CdII forms reveal the presence of CdII(S-Cys)3{O(H)-Ser} centers in the surface ligand mutants C9S and C42S by the detection of 113Cd−O−CHβ 2 coupling and S−OHγ resonances. The assumption of the presence of FeII(S-Cys)3(O-Ser) centers in each mutant protein at pH values above the characteristic pK a red allows a simple interpretation of the electrochemical behavior. Protonation of the Fe−Oγ-Ser link upon reduction is proposed, followed by hydrolysis at lower pH values:  FeIII−Oγ-Ser + H+ + e- → FeII−Oγ(H)-Ser; FeII−Oγ(H)-Ser + H2O → FeII−OH2 + HOγ-Ser. The differences in reduction potentials, their pH dependence, and the onset of irreversible electrochemistry can be attributed to differences in the Fe−O bonds of the interior and surface ligands. These differences appear to result from variation in the conformational flexibility of the protein chelate loops which carry the ligands. An attempt to generate crystals of the reduced FeII-C42S protein by treatment of FeIII-C42S crystals with dithionite at pH 4 led to loss of iron. A crystal structure (1.6 Å; R, 16.8%) reveals that cysteine residues 6 and 9 have trapped the oxidation product SO2, a result confirmed by reactions in solution:  Cys-SH + SO2 → Cys-SII−SIVO2 - + H+.

CVF is a complex body fluid consisting of both endogenous and environmental proteins. The putative role of some of these proteins in the human reproductive tract is discussed.

Conserved glycine residues at positions 10 and 43 in the electron transfer protein rubredoxin (active site: Fe-(Cys-S) 4 ) from Clostridium pasteurianum are related by a pseudo-2-fold symmetry. They have been mutated to alanine and valine and four single and two double mutant (G10V/G43A and G10V/G43V) proteins expressed in stable form in Escherichia coli. Physical properties were modified by steric interactions between the βand γ-carbon substituents of the new side chains and the CO functions of C9 and C42 and other adjacent groups. These interactions perturb the chelate loops formed by residues 5-11 and 38-44. 1 H NMR results for Cd(II) forms indicate that the Pr i side chain of V10 in the G10V mutant occupies the surface pocket defined by loop 5-11 and thereby modifies the environment of the 5-11 NH protons. The equivalent side chain of V43 in G43V is denied the same access to the 38-44 pocket. This leads to a specific perturbation of the V44-NH‚‚‚S-C42 interaction in this mutant. These effects are additive in the double mutant G10V/G43V, consistent with the different structural changes being localized in each loop. The midpoint potentials of the iron forms of the six mutants are shifted negatively relative to the recombinant protein by -16 to -86 mV. A G f V mutation has a larger effect than a G f A, but again, an additivity of the differential effects is seen in the double mutants. Minor perturbations of resonance Raman and electronic spectra are dominated by the mutation at G10. Overall, the present work represents one approach to the systematic exploration of the influence of the protein chain upon the fundamental properties of this molecule.

BackgroundC-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentameric protein in plasma. Recently, a potential dissociation mechanism from the disc-shaped pentameric CRP (pCRP) into single monomers (monomeric or mCRP) has been described. It has been shown that mCRP has strong pro-inflammatory effects on monocytes. To further define the role of mCRP in determining monocyte phenotype, the effects of CRP isoforms on THP-1 protein expression profiles were determined. The hypothesis to be tested was that mCRP induces specific changes in the protein expression profile of THP-1 cells that differ from that of pCRP.MethodsProtein cell lysates from control and mCRP, pCRP or LPS-treated THP-1 cells were displayed using 2-dimensional SDS PAGE and compared. Differentially expressed proteins were identified by MALDI-TOF MS and confirmed by Western blotting.ResultsmCRP significantly up-regulates ubiquitin-activating enzyme E1, a member of the ubiquitin-proteasome system in THP-1 monocytes. Furthermore, HSP 70, alpha-actinin-4 (ACTN4) and alpha-enolase/enolase 1 were upregulated. The proteomic profile of LPS and pCRP treated monocytes differ significantly from that of mCRP.ConclusionThe data obtained in this study support the hypothesis that isoform-specific effects of CRP may differentially regulate the phenotype of monocytes.