The transfer of electrons between proteins is an essential step in biological energy production. Two protein redox partners are often artificially crosslinked to investigate the poorly understood mechanism by which they interact. To better understand the effect of crosslinking on electron transfer rates, we have constructed dimers of azurin by crosslinking the monomers. The measured electron exchange rates, combined with crystal structures of the dimers, demonstrate that the length of the linker can have a dramatic effect on the structure of the dimer and the electron transfer rate. The presence of ordered water molecules in the protein-protein interface may considerably influence the electronic coupling between redox centers.
Retinoid-binding proteins play an important role in regulating transport, storage, and metabolism of vitamin A and its derivatives. The solution structure and backbone dynamics of rat cellular retinol-binding protein type I (CRBP) in the apo-and holo-form have been determined and compared using multidimensional high resolution NMR spectroscopy. The global fold of the protein is consistent with the common motif described for members of the intracellular lipid-binding protein family. The most relevant difference between the NMR structure ensembles of apo-and holoCRBP is the higher backbone disorder, in the ligand-free form, of some segments that frame the putative entrance to the ligandbinding site. These comprise ␣-helix II, the subsequent linker to -strand B, the hairpin turn between -strands C and D, and the E-F turn. The internal backbone dynamics, obtained from 15 N relaxation data (T 1 , T 2 , and heteronuclear nuclear Overhauser effect) at two different fields, indicate several regions with significantly higher backbone mobility in the apoprotein, including the C-D and E-F turns. Although apoCRBP contains a binding cavity more shielded than that of any other retinoid carrier, conformational flexibility in the portal region may assist retinol uptake. The stiffening of the backbone in the holoprotein guarantees the stability of the complex during retinol transport and suggests that targeted retinol release requires a transiently open state that is likely to be promoted by the acceptor or the local environment.Vitamin A derivatives play important roles in a variety of biological processes including vision, cell growth, cell differentiation, and morphogenesis (1). Plasma transport of retinol to target cells and intracellular transport for either storage or metabolic conversion are performed by binding proteins that belong to the calycin superfamily. The cytosolic carriers are members of the intracellular lipid-binding protein (i-LBP) 1 family, characterized by molecular masses of around 15 kDa. Their structure consists of a 10-stranded -barrel, formed by two orthogonal -sheets, and two short ␣-helices (2). The two best known intracellular carriers of retinol are cellular retinolbinding protein type I (CRBP), widely distributed in various tissues (3, 4), and cellular retinol-binding protein type II (CRBP-II), present in the enterocytes of the small intestine and in neonatal hepatocytes (5, 6). The structures of rat apo-and holoCRBP-II have been solved both in the crystal (7) and in solution (8, 9), whereas the only structure of CRBP available to date was that of the holoprotein in the crystal (10). More recently, two other retinol carriers have been identified as follows: murine CRBP-III, expressed primarily in heart, muscle, and adipose tissue (11); and human CRBP-III, most abundant in liver and kidney, whose structure in the retinol-free form has been solved by x-ray crystallography (12).In the cell, the poorly water-soluble retinol is stored within membranes as a retinyl-ester derivative of long-cha...
Flavodoxin II from Azotobacter vinelandii is a ''long-chain'' flavodoxin and has one of the lowest E 1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 Å by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five-stranded parallel b-sheet flanked on either side by a-helices. An eight-residue insertion, compared with other long-chain flavodoxins, forms a short 3 10 helix preceding the start of the a 3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent-accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E 2 value in particular.Keywords: flavodoxin; FMN; hydrogen bonding; polarity; redox potentialsThe redox potential of an electron transfer protein is of prime importance in relation to its function, where the correlation between protein structure and redox potential helps explain how nature has adapted proteins to their specific functions. In the past, this has been studied for a range of flavodoxins, small (14-23 kDa) acidic a/b proteins that contain a single noncovalently bound flavin mononucleotide (FMN) cofactor, from different organisms. In vivo they act as remarkably versatile low potential one-electron donors in a range of reactions (Mayhew and Tollin 1992) in mainly prokaryotic organisms, including obligate and facultative anaerobes, microaerophiles, and photosynthetic cyanobacteria, as well as in both red and green eukaryotic algae. In the photosynthetic bac-
Vaccines targeting the human papillomavirus (HPV) minor capsid protein L2 are emerging as chemico-physically robust and broadly protective alternatives to the current HPV (L1-VLP) vaccines. We have previously developed a trivalent L2 vaccine prototype exploiting Pyrococcus furiosus thioredoxin (PfTrx) as a thermostable scaffold for the separate presentation of three distinct HPV L2(20–38) epitopes. With the aim of achieving a highly immunogenic, yet simpler and more GMP-production affordable formulation, we report here on a novel thermostable nanoparticle vaccine relying on genetic fusion of PfTrx-L2 with the heptamerizing coiled-coil polypeptide OVX313. A prototype HPV16 monoepitope version of this nanoparticle vaccine (PfTrx-L2-OVX313; median radius: 8.6 ± 1.0 nm) proved to be approximately 10-fold more immunogenic and with a strikingly enhanced cross-neutralization capacity compared to its monomeric counterpart. Vaccine-induced (cross-)neutralizing responses were further potentiated in a multiepitope derivative displaying eight different L2(20–38) epitopes, which elicited neutralizing antibodies against 10 different HPVs including three viral types not represented in the vaccine. Considering the prospective safety of the PfTrx scaffold and of the OVX313 heptamerization module, PfTrx-OVX313 nanoparticles lend themselves as robust L2-based immunogens with a high translational potential as a 3rd generation HPV vaccine, but also as a novel and extremely versatile peptide-antigen presentation platform.
A 95 GHz pulsed deuterium ENDOR study has been performed on single crystals of azurin from Pseudomonas aeruginosa selectively deuterated at the C(beta) position of the copper-coordinating cysteine 112. Complete hyperfine tensors of the two deuterium atoms have been obtained, which reveal identical isotropic parts. Analysis of the hyperfine tensors provides insight into the spin-density delocalization over the cysteine ligand. Approximately 45 % of the spin density in the paramagnetic site can be attributed to copper and 30 % to sulfur.
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