Computer-assisted mass spectrometric analysis of naturally occurring and artificially introduced cross-links in proteins and protein complexes

A versatile software tool, VIRTUALMSLAB, is presented that can perform advanced complex virtual proteomic experiments with mass spectrometric analyses to assist in the characterization of proteins. The virtual experimental results allow rapid, flexible and convenient exploration of sample preparation strategies and are used to generate MS reference databases that can be matched with the real MS data obtained from the equivalent real experiments. Matches between virtual and acquired data reveal the identity and nature of reaction products that may lead to characterization of post-translational modification patterns, disulfide bond structures, and cross-linking in proteins or protein complexes. The most important unique feature of this program is the ability to perform multistage experiments in any user-defined order, thus allowing the researcher to vary experimental approaches that can be conducted in the laboratory. Several features of VIRTUALMSLAB are demonstrated by mapping both disulfide bonds and artificially introduced protein cross-links. It is shown that chemical cleavage at aspartate residues in the protease resistant RNase A, followed by tryptic digestion can be optimized so that the rigid protein breaks up into MALDI-MS detectable fragments, leaving the disulfide bonds intact. We also show the mapping of a number of chemically introduced cross-links in the NK1 domain of hepatocyte growth factor/scatter factor. The VIRTUALMSLAB program was used to explore the limitation and potential of mass spectrometry for cross-link studies of more complex biological assemblies, showing the value of high performance instruments such as a Fourier transform mass spectrometer. The program is freely available upon request.     

UCP1-independent thermogenesis in white adipose tissue of cold-acclimated Ucp1-/- mice

Apart from UCP1-based nonshivering thermogenesis in brown adipocytes, the identity of thermogenic mechanisms that can be activated to reduce a positive energy balance is largely unknown. To identify potentially useful mechanisms, we have analyzed physiological and molecular mechanisms that enable mice, genetically deficient in UCP1 and sensitive to acute exposure to the cold at 4 degrees C, to adapt to long term exposure at 4 degrees C. UCP1-deficient mice that can adapt to the cold have increased oxygen consumption and show increased oxidation of both fat and glucose as indicated from serum metabolite levels and liver glycogen content. Enhanced energy metabolism in inguinal fat was also indicated by increased oxygen consumption and fat oxidation in tissue suspensions and increased AMP kinase activity in dissected tissues. Analysis of gene expression in skeletal muscle showed surprisingly little change between cold-adapted Ucp1+/+ and Ucp1-/- mice, whereas in inguinal fat a robust induction occurred for type 2 deiodinase, sarcoendoplasmic reticulum Ca2+-ATPase, mitochondrial glycerol 3-phosphate dehydrogenase, PGC1alpha, CoxII, and mitochondrial DNA content. Western blot analysis showed an induction of total phospholamban and its phosphorylated form in inguinal fat and other white fat depots, but no induction was apparent in muscle. We conclude that alternative thermogenic mechanisms, based in part upon the enhanced capacity for ion and substrate cycling associated with brown adipocytes in white fat depots, are induced in UCP1-deficient mice by gradual cold adaptation.     

Reconstitution of the basal calcium transport in resealed human red blood cell ghosts

The (45)Ca(2+) influx into right-side-out resealed ghosts (RG) prepared from human red blood cells (RBC) was measured. The (45)Ca(2+) equilibration occurred with t(1/2)=2.5 min and the steady-state was reached after 17 min with the level of 22+/-2 micromol/L(packed cells) at 37 degrees C. The rate of the influx was 97+/-17 micromol/L(packed cells)h. The (45)Ca(2+) influx was saturated with [Ca(2+)](0) at 4 mmol/L and was optimal at pH 6.5 and 30 degrees C. Divalent cations (10(-4)-10(-6)mol/L), nifedipine (10(-5)-10(-4)mol/L), DIDS (up to 10(-4)mol/L), and quinidine (10(-4)-10(-3)mol/L), inhibited the (45)Ca(2+) influx while uncoupler (10(-6)-10(-5)mol/L) stimulated it. In contrast to intact RBC, vanadate inhibited the (45)Ca(2+) influx when added to the external medium, however, the stimulation was observed when vanadate was present in media during both lysis and resealing. PMA had no effect under conditions found to stimulate the Ca(2+) influx in intact RBC. The results show that the Ca(2+) influx into RG is a carrier-mediated process but without control by protein kinase C and that the influx and efflux of Ca(2+) are coupled via the H(+) homeostasis similarly as in intact RBC but with modified mechanism.     

A challenging insight on the structural unit 1 of molluscan Rapana venosa hemocyanin

Hemocyanins of mollusks are high molecular mass glycoproteins with a complex quaternary structure which still remains to be defined in detail for most of its species as far as number, spatial distribution and interactions of their structural units is concerned. In the present study, we isolated the functional units of the structural subunit RvH1 of Rapana venosa hemocyanin, combining enzymatic and non-enzymatic methods. Our results suggest that Hc's carbohydrate moieties play a basic role in the organization of the structural units, resulting from post-translational polymerization of the 50 kDa functional units and involving sugar moieties that link between them.     

Sequence analysis of the complete mitochondrial DNA in 10 commonly used inbred rat strains

Rat remains a major biomedical model system for common, complex diseases. The rat continues to gain importance as a model system with the completion of its full genomic sequence. Although the genomic sequence has generated much interest, only three complete sequences of the rat mitochondria exist. Therefore, to increase the knowledge of the rat genome, the entire mitochondrial genomes (16,307–16,315 bp) from 10 inbred rat strains (that are standard laboratory models around the world) and 2 wild rat strains were sequenced. We observed a total of 195 polymorphisms, 32 of which created an amino acid change (nonsynonymous substitutions) in 12 of the 13 protein coding genes within the mitochondrial genome. There were 11 single nucleotide polymorphisms within the tRNA genes, six in the 12S rRNA, and 12 in the 16S rRNA including 3 insertions/deletions. We found 14 single nucleotide polymorphisms and 2 insertion/deletion polymorphisms in the D-loop. The inbred rat strains cluster phylogenetically into three distinct groups. The wild rat from Tokyo grouped closely with five inbred strains in the phylogeny, whereas the wild rat from Milwaukee was not closely related to any inbred strain. These data will enable investigators to rapidly assess the potential impact of the mitochondria in these rats on the physiology and the pathophysiology of phenotypes studied in these strains. Moreover, these data provide information that may be useful as new animal models, which result in novel combinations of nuclear and mitochondrial genomes, are developed. genome; mitochondria     

UreG, a chaperone in the urease assembly process, is an intrinsically unstructured GTPase that specifically binds Zn2+

Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was overexpressed in Escherichia coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing, and mass spectrometry. A combination of size exclusion chromatography and multiangle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), whereas NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a kcat=0.04 min(-1), confirming a role for this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds two Zn2+ ions per dimer, with a KD=42 +/- 3 microm, and has a 10-fold lower affinity for Ni2+. A structural model for BpUreG was calculated by using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open beta-barrel surrounded by alpha-helices and a P-loop at the N terminus. The protein dynamic behavior observed in solution is critically discussed relative to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins that need the interaction with cofactors or other protein partners to perform their function. It is also proposed that metal ions such as Zn2+ could have important structural roles in the urease activation process.     

Structure–function relationships of four compression wood types: micromechanical properties at the tissue and fibre level

The mechanisms behind compressive stress generation in gymnosperms are not yet fully understood. Investigating the structure–function relationships at the tissue and cell level, however, can provide new insights. Severe compression wood of all species lacks a S3 layer, has a high microfibril angle in the S2 layer and a high lignin content. Additionally, special features like helical cavities or spiral thickenings appear, which are not well understood in terms of their mechanical relevance, but need to be examined with regard to evolutionary trends in compression wood development. Thin compression wood foils and isolated tracheids of four gymnosperm species [Ginkgo biloba L., Taxus baccata L., Juniperus virginiana L., Picea abies (L.) Karst.] were investigated. The tracheids were isolated mechanically by peeling them out of the solid wood using fine tweezers. In contrast to chemical macerations, the cell wall components remained in their original condition. Tensile properties of tissue foils and tracheids were measured in a microtensile apparatus under wet conditions. Our results clearly show an evolutionary trend to a much more flexible compression wood. An interpretation with respect to compressive stress generation is discussed.