Biomedical subjects

Patricio O Craig

Publications and source records attributed to Patricio O Craig.

4 recordsLinked to original sources

PubMed2006

Exploring protein interfaces with a general photochemical reagent.

Protein folding, natural conformational changes, or interaction between partners involved in recognition phenomena brings about differences in the solvent-accessible surface area (SASA) of the polypeptide chain. This primary event can be monitored by the differential chemical reactivity of functional groups along the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generates methylene carbene (:CH(2)). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. (3)H-DZN was successfully used in our laboratory for studying protein structure and folding. Here we address for the first time the usefulness of this probe to examine the area of interaction in protein-protein complexes. For this purpose we chose the complex formed between hen egg white lysozyme (HEWL) and the monoclonal antibody IgG(1) D1.3. :CH(2) labeling of free HEWL or complexed with IgG(1) D1.3 yields 2.76 and 2.32 mmol CH(2) per mole protein at 1 mM DZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrement in the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), in agreement with the known unspecific surface labeling reaction of :CH(2). Further comparative analysis at the level of tryptic peptides led to the identification of the sites involved in the interaction. Remarkably, those peptides implicated in the contact area show the highest differential labeling: H(15)GLDNYR(21), G(117)TDVQAWIR(125), andG(22)YSLGNWVCAAK(33). Thus, protein footprinting with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.

Amino Acid Sequence↗

PubMed2004

Bacillus subtilis DesR functions as a phosphorylation-activated switch to control membrane lipid fluidity.

The Des pathway of Bacillus subtilis regulates the synthesis of the cold-shock induced membrane-bound enzyme Delta5-fatty acid desaturase (Delta5-Des). A central component of the Des pathway is the response regulator, DesR, which is activated by a membrane-associated kinase, DesK, in response to a decrease in membrane lipid fluidity. Despite genetic and biochemical studies, specific details of the interaction between DesR and the DNA remain unknown. In this study we show that only the phosphorylated form of protein DesR is able to bind to a regulatory region immediately upstream of the promoter of the Delta5-Des gene (Pdes). Phosphorylation of the regulatory domain of dimeric DesR promotes, in a cooperative fashion, the hierarchical occupation of two adjacent, non-identical, DesR-P DNA binding sites, so that there is a shift in the equilibrium toward the tetrameric active form of the response regulator. Subsequently, this phosphorylation signal propagation leads to the activation of the des gene through recruitment of RNA polymerase to Pdes. This is the first dissected example of a transcription factor functioning as a phosphorylation-activated switch for a cold-shock gene, allowing the cell to optimize the fluidity of membrane phospholipids.

Bacillus subtilis↗

PubMed2003

High order quaternary arrangement confers increased structural stability to Brucella sp. lumazine synthase.

The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18-kDa monomers, each extensively contacting neighboring monomers. The icosahedrical structure consists of 60 LS monomers arranged as 12 pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella sp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedric enzymes. This unusual divergence prompted us to further investigate its quaternary arrangement. In the present work, we demonstrate by means of solution light scattering and x-ray structural analyses that BLS assembles as a very stable dimer of pentamers, representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 degrees C compared with pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins.

Binding Sites↗

PubMed2002

Probing protein conformation with a minimal photochemical reagent.

3H-diazirine (3H-DZN), a photoreactive gas similar in size to water, was used to probe the topography of the surface and inner space of proteins. On photolysis 3H-DZN generates 3H-methylene carbene, which reacts unselectively with its molecular cage, inserting even into C-H bonds. Labeling of bovine alpha-lactalbumin (alpha-LA, MW: 14,200) with 1 mM (3)H-DZN yielded 0.0041 mol CH2/mol of protein, in agreement with the expectation for an unspecific surface-labeling phenomenon. The cooperative urea-induced unfolding of alpha-LA, as monitored by the extent of 3H-methylene labeling, agrees with that measured by circular dichroism spectroscopy in the far and near ultraviolet regions. At 8 M urea, the unfolded state U was labeled 25-30% more than the native state N primarily because of the increase in the accessible surface area (ASA) of the protein occurring upon unfolding. However, this result lies below the approximately 100% increment expected from theoretical estimates of ASA of state U. Among other factors, most likely the existence of a residual structure in U, that involves helices H2 and H4 of the alpha subdomain, might account for this fact, as shown by a comparative analysis of peptide labeling patterns of N and U samples. In this paper, we demonstrate the usefulness of the 3H-methylene labeling method to monitor conformational transitions and map solvent accessibility along the polypeptide sequence, thus opening the possibility of outlining structural features of nonnative states (i.e., denatured states, molten globule). We anticipate that this technique also would help to identify ligand binding and oligomerization sites in proteins.

Animals↗