A prokaryotic protein, YtvA from Bacillus subtilis, was found to possess a light, oxygen, voltage (LOV) domain sharing high homology with the photoactive, flavin mononucleotide (FMN)-binding LOV domains of phototropins (phot), blue-light photoreceptors for phototropism in higher plants. Computer-based three-dimensional modeling suggests that YtvA-LOV binds FMN in a similar pocket as phot-LOVs. Recombinant YtvA indeed exhibits the same spectroscopical features and blue-light-induced photochemistry as phot-LOVs, with the reversible formation of a blue-shifted photoproduct, assigned to an FMN-cysteine thiol adduct (Thio 383 ). By means of laser-flash photolysis and time-resolved optoacoustic experiments, we measured the quantum yield of formation for Thio 383 , ⌽ Thio ϭ 0.49, and the enthalpy change, ⌬H Thio ϭ 135 kJ/mol, with respect to the parent state. The formation of Thio 383 is accompanied by a considerable volume contraction, ⌬V Thio ϭ Ϫ13.5 ml/mol. Similar to phot-LOVs, Thio 383 is formed from the decay of a red-shifted transient species, T 650 , within 2 s. In both YtvA and free FMN, this transient has an enthalpy content of ϳ200 kJ/mol, and its formation is accompanied by a small contraction, ⌬V T Ϸ Ϫ1.5 ml/mol, supporting the assignment of T 650 to the FMN triplet state, as suggested by spectroscopical evidences. These are the first studies indicating that phototropin-related, blue-light receptors may exist also in prokaryotes, besides constituting a steadily growing family in plants.

The myelin sheath is a tightly packed multilayered membrane structure insulating selected axons in the central and the peripheral nervous systems. Myelin is a biochemically unique membrane, containing a specific set of proteins. In this study, we expressed and purified recombinant human myelin P2 protein and determined its crystal structure to a resolution of 1.85 Å. A fatty acid molecule, modeled as palmitate based on the electron density, was bound inside the barrel-shaped protein. Solution studies using synchrotron radiation indicate that the crystal structure is similar to the structure of the protein in solution. Docking experiments using the high-resolution crystal structure identified cholesterol, one of the most abundant lipids in myelin, as a possible ligand for P2, a hypothesis that was proven by fluorescence spectroscopy. In addition, electrostatic potential surface calculations supported a structural role for P2 inside the myelin membrane. The potential membrane-binding properties of P2 and a peptide derived from its N terminus were studied. Our results provide an enhanced view into the structure and function of the P2 protein from human myelin, which is able to bind both monomeric lipids inside its cavity and membrane surfaces.

The role of calcium ions in the regulation of tissue transglutaminase is investigated by experimental approaches and computer modeling. A three-dimensional model of the transglutaminase is computed by homology building on crystallized human factor XIII and is used to interpret structural and functional results. The molecule is a prolate ellipsoid (6.2 Â 4.2 Â 11 nm) and comprises four domains, assembled pairwise into N-terminal and C-terminal regions. The active site is hidden in a cleft between these regions and is inaccessible to macromolecular substrates in the calcium-free form. Protein dynamics simulation indicates that these regions move apart upon addition of calcium ions, revealing the active site for catalysis.The protein dimensions are consistent with results obtained with small-angle neutron and X-ray scattering. The gyration radius of the protein (3 nm) increases in the presence of calcium ions (3.9 nm), but it is virtually unaffected in the presence of GTP, suggesting that only calcium ions can promote major structural changes in the native protein.Proteolysis of an exposed loop connecting the N-terminal and C-terminal regions is linearly correlated with enzyme inactivation and prevents the calcium-induced conformational changes.

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and with cytoskeletal and other proteins. A central segment of MBP is highly conserved in mammals and consists of a membrane surface-associated amphipathic alpha-helix, immediately followed by a proline-rich segment that we hypothesize is an SH3 ligand. We show by circular dichroic spectroscopy that this proline-rich segment forms a polyproline type II helix in vitro under physiological conditions and that phosphorylation at a constituent threonyl residue has a stabilizing effect on its conformation. Using SH3 domain microarrays, we observe that the unmodified recombinant murine 18.5 kDa MBP isoform (rmC1 component) binds the following SH3 domains: Yes1 > PSD95 > cortactin = PexD = Abl = Fyn = c-Src = Itk in order of decreasing affinity. A quasi-deiminated form of the protein (rmC8) binds the SH3 domains Yes1 > Fyn > cortactin = c-Src > PexD = Abl. Phosphorylation of rmC1 at 1-2 threonines within the proline-rich segment by mitogen-activated protein kinase in vitro has no effect on the binding specificity to the SH3 domains on the array. An SH3 domain of chicken Fyn is also demonstrated to bind to lipid membrane-associated C1, phosphorylated C1, and rmC8. Molecular docking simulations of the interaction of the putative SH3 ligand of classic MBP with the human Fyn SH3 domain indicate that the strength of the interaction is of the same order of magnitude as with calmodulin and that the molecular recognition and association is mediated by some weak CH...pi interactions between the ligand prolyl residues and the aromatic ones of the SH3 binding site. One such interaction is well-conserved and involves the stacking of an MBP-peptide prolyl and an SH3 domain tryptophanyl residue, as in most other SH3-ligand complexes. Lysyl and arginyl residues in the peptide canonically interact via salt bridges and cation-pi interactions with negatively charged and aromatic residues in the SH3 domain binding site. Posttranslational modifications (phosphorylation or methylation) of the ligand cause noticeable shifts in the conformation of the flexible peptide and its side chains but do not predict any major inhibition of the binding beyond somewhat less favorable interactions for peptides with phosphorylated seryl or threonyl residues.