Photochemical and electrophysical production of radicals on millisecond timescales to probe the structure, dynamics and interactions of proteins.

The reaction of hydroxyl and other oxygen-based radicals with the side chains of proteins on millisecond timescales has been used to probe the structure of proteins, their dynamics in solution and interactions with other macromolecules. Radicals are generated in high flux within microseconds from synchrotron radiation and discharge sources and react with proteins on timescales that are less than those often attributed to structural reorganisation and folding. The oxygen-based radicals generated in aqueous solution react with proteins to effect limited oxidation at specific amino acids throughout the sequence of the protein. The extent of oxidation at these residue markers is highly influenced by the accessibility of the reaction site to the bulk solvent. The extent of oxidation allows protection levels to be measured based on the degree to which a reaction occurs. A map of a protein's three-dimensional structure is subsequently assembled as in a footprinting experiment. Protein solutions that contain various concentrations of substrates that either promote or disrupt structural transitions can be investigated to facilitate site-specific equilibrium and time-resolved studies of protein folding. The radical-based strategies can also be employed in the study of protein-protein interactions to provide a new avenue for investigating protein complexes and assemblies with high structural resolution. The urea-induced unfolding of apomyoglobin, and the binding domains within the ribonuclease S and calmodulin-melittin protein-peptide complexes are presented to illustrate the approach.     

Song is a reliable signal of bill morphology in Darwin's small tree finch Camarhynchus parvulus, and vocal performance predicts male pairing success

The shape and movement of the vocal tract are known to influence bird song. Current theory predicts that large bill and body size are correlated with low frequency song and slow trill rate. It is also widely accepted that song characteristics are important for mate choice by females. We investigated the relationship between bill morphology, song characteristics, and pairing success in Darwin's small tree finch Camarhynchus parvulus , on the Galapagos Islands. Contrary to predictions from a previous cross-species study on Darwin's finches, we found that individuals with larger bill size produced songs with slow trill rate, high dominant frequency, and broad frequency bandwidth, indicating that song is a reliable signal of bill morphology. Vocal performance as indicated by the deviation from an upper performance limit was higher in paired than unpaired males. Pairing was not skewed in favour of a particular bill size, and both small and large billed males that sang high performance song had high pairing success. The reliable signalling function of song has implications for female choice and territorial defence, given that both females and conspecific competitors can assess the relative size of males' bills through song, while females may use vocal performance as a signal of male quality.     

Role of biotin-containing membranes and nuclear distribution in differentiating human endometrial cells

Human Ishikawa endometrial cells form domes when confluent monolayers are stimulated with fresh fetal bovine serum. Extensive structural and biochemical changes have been detected during the approximately 30 h differentiation period. The earliest detectable change involves the formation of multinucleated structures and the appearance of “granules” that stain for biotin within those structures. Nuclei become associated with each other and are ultimately enclosed within a biotin-containing membrane. Aggregated membrane-sheathed nuclei and the cells containing them begin to elevate from the dish as biotin staining becomes apparent in apical membranes. The elevated structures are called predomes and consist of one or more very large cells containing the sheathed nuclei. Apical membranes of these unusual cells extend far out into the medium in structures that resemble endometrial pinopods. A lumen under the elevated cells fills with transcytosed fluid. As differentiation proceeds, highly concentrated chromatin material that was flattened against apical and lateral membranes of the predome cells begins to disperse. Small mononuclear cells evolve from larger predome cells. Apical membranes of predome and dome cells continue to stain for biotin. Gel electrophoresis of SDS-solubilized biotin-containing membranes, followed by Western blot analysis using avidin-linked peroxidase, resulted in three stained bands with molecular weights similar to those of the mitochondrial carboxylases: propionyl carboxylase, methylmalonyl carboxylase, and pyruvate carboxylase. J. Cell. Biochem. 71:400–415, 1998. © 1998 Wiley-Liss, Inc.     

Genetic interactions of smc, ftsK, and parB genes in Streptomyces coelicolor and their developmental genome segregation phenotypes

The mechanisms by which chromosomes condense and segregate during developmentally regulated cell division are of interest for Streptomyces coelicolor, a sporulating, filamentous bacterium with a large, linear genome. These processes coordinately occur as many septa synchronously form in syncytial aerial hyphae such that prespore compartments accurately receive chromosome copies. Our genetic approach analyzed mutants for ftsK, smc, and parB. DNA motor protein FtsK/SpoIIIE coordinates chromosome segregation with septum closure in rod-shaped bacteria. SMC (structural maintenance of chromosomes) participates in condensation and organization of the nucleoid. ParB/Spo0J partitions the origin of replication using a nucleoprotein complex, assembled at a centromere-like sequence. Consistent with previous work, we show that an ftsK-null mutant produces anucleate spores at the same frequency as the wild-type strain (0.8%). We report that the smc and ftsK deletion-insertion mutants (ftsK′ truncation allele) have developmental segregation defects (7% and 15% anucleate spores, respectively). By use of these latter mutants, viable double and triple mutants were isolated in all combinations with a previously described parB-null mutant (12% anucleate spores). parB and smc were in separate segregation pathways; the loss of both exacerbates the segregation defect (24% anucleate spores). For a triple mutant, deletion of the region encoding the FtsK motor domain and one transmembrane segment partially alleviates the segregation defect of the smc parB mutant (10% anucleate spores). Considerable redundancy must exist in this filamentous organism because segregation of some genomic material occurs 90% of the time during development in the absence of three functions with only a fourfold loss of spore viability. Furthermore, we report that scpA and scpAB mutants (encoding SMC-associated proteins) have spore nucleoid organization defects. Finally, FtsK-enhanced green fluorescent protein (EGFP) localized as bands or foci between incipient nucleoids, while SMC-EGFP foci were not uniformly positioned along aerial hyphae, nor were they associated with every condensing nucleoid.