Differential telomere processing by Borrelia telomere resolvases in vitro but not in vivo

Causative agents of Lyme disease and relapsing fever, including Borrelia burgdorferi and Borrelia hermsii, respectively, are unusual among bacteria in that they possess a segmented genome with linear DNA molecules terminated by hairpin ends, known as telomeres. During replication, these telomeres are processed by the essential telomere resolvase, ResT, in a unique biochemical reaction known as telomere resolution. In this study, we report the identification of the B. hermsii resT gene through cross-species hybridization. Sequence comparison of the B. hermsii protein with the B. burgdorferi orthologue revealed 67% identity, including all the regions currently known to be crucial for telomere resolution. In vitro studies, however, indicated that B. hermsii ResT was unable to process a replicated B. burgdorferi type 2 telomere substrate. In contrast, in vivo cross-species complementation in which the native resT gene of B. burgdorferi was replaced with B. hermsii resT had no discernible effect, even though B. burgdorferi strain B31 carries at least two type 2 telomere ends. The B. burgdorferi ResT protein was also able to process two telomere spacing mutants in vivo that were unresolvable in vitro. The unexpected differential telomere processing in vivo versus in vitro by the two telomere resolvases suggests the presence of one or more accessory factors in vivo that are normally involved in the reaction. Our current results are also expected to facilitate further studies into ResT structure and function, including possible interaction with other Borrelia proteins.     

Diffusional encounter of barnase and barstar

We present an analysis of trajectories from Brownian dynamics simulations of diffusional protein-protein encounter for the well-studied system of barnase and barstar. This analysis reveals details about the optimal association pathways, the regions of the encounter complex, possible differences of the pathways for dissociation and association, the coupling of translational and rotation motion, and the effect of mutations on the trajectories. We found that a small free-energy barrier divides the energetically most favorable region into a region of the encounter complex above the barnase binding interface and a region around a second energy minimum near the RNA binding loop. When entering the region of the encounter complex from the region near the RNA binding loop, barstar has to change its orientation to increase the electrostatic attraction between the proteins. By concentrating the analysis on the successful binding trajectories, we found that the region of the second minimum is not essential for the binding of barstar to barnase. Nevertheless, this region may be helpful to steer barstar into the region of the encounter complex. When applying the same analysis to several barnase mutants, we found that single mutations may drastically change the free-energy landscape and may significantly alter the population of the two minima. Therefore, certain protein-protein pairs may require careful adaptation of the positions of encounter and transition states when interpreting mutation effects on kinetic rates of association and/or dissociation.     

Brønsted analysis reveals Lys218 as the carboxylase active site base that deprotonates vitamin K hydroquinone to initiate vitamin K-dependent protein carboxylation

The vitamin K-dependent (VKD) carboxylase converts Glu's to carboxylated Glu's in VKD proteins to render them functional in a broad range of physiologies. The carboxylase uses vitamin K hydroquinone (KH(2)) epoxidation to drive Glu carboxylation, and one of its critical roles is to provide a catalytic base that deprotonates KH(2) to allow epoxidation. A long-standing model invoked Cys as the catalytic base but was ruled out by activity retention in a mutant where every Cys is substituted by Ala. Inhibitor analysis of the cysteine-less mutant suggested that the base is an activated amine [Rishavy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13732-13737], and in the present study, we used an evolutionary approach to identify candidate amines, which revealed His160, His287, His381, and Lys218. When mutational analysis was performed using an expression system lacking endogenous carboxylase, the His to Ala mutants all showed full epoxidase activity but K218A activity was not detectable. The addition of exogenous amines restored K218A activity while having little effect on wild type carboxylase, and pH studies indicated that rescue was dependent upon the basic form of the amine. Importantly, Br?nsted analysis that measured the effect of amines with different pK(a) values showed that K218A activity rescue depended upon the basicity of the amine. The combined results provide strong evidence that Lys218 is the essential base that deprotonates KH(2) to initiate the reaction. The identification of this base is an important advance in defining the carboxylase active site and has implications regarding carboxylase membrane topology and the feedback mechanism by which the Glu substrate regulates KH(2) oxygenation.     

Characterization of the linker 2 region in human vimentin using site-directed spin labeling and electron paramagnetic resonance

Site-directed spin labeling and electron paramagnetic resonance were used to probe residues 281-304 of human vimentin, a region that has been predicted to be a non-alpha-helical linker and the beginning of coiled-coil domain 2B. Though no direct test of linker structure has ever been made, this region has been hypothesized to be flexible with the polypeptide chains looping away from one another. EPR analysis of spin-labeled mutants indicates that (a) several residues reside in close proximity, suggesting that adjacent linker regions in a dimer run in parallel, and that (b) the polypeptide backbone is relatively rigid and inflexible in this region. However, this region does not show the characteristics of a coiled-coil as has been identified elsewhere in the molecule. Within this region, spectra from positions 283 and 291 are unique from all others thus far examined. These positions, predicted to be in a noncoiled-coil structure, display a significantly stronger interaction than the a-d contact positions of coiled-coil regions. Analysis of the early stages of assembly by dialysis from 8 M urea and progressive thermal denaturation shows the close apposition and structural rigidity at residues 283 and 291 occurs very early in assembly and with a relatively sudden onset, well before coiled-coil formation in other parts of the molecule. These features are inconsistent with hypotheses that envision the linkers as flexible regions, or as looping away from one another, and raise the possibility that the linker may be the site at which dimer alignment and/or formation is initiated. Spin labels placed further downstream yield spectra suggesting that the first regular heptad of rod domain 2 begins at position 302. In conjunction with our previous characterization of region 305-336 and the solved structure of rod 2B from 328-405, the full extent of coiled-coil domain in rod 2B is now known, spanning from vimentin positions 302-405.     

Death-associated protein kinase phosphorylates mammalian ribosomal protein S6 and reduces protein synthesis

Death-associated protein kinase (DAPK) is a pro-apoptotic, calcium/calmodulin-regulated protein kinase that is a drug discovery target for neurodegenerative disorders. Despite the potential profound physiological role of DAPK in neuronal function and pathophysiology, the endogenous substrate(s) of this kinase and the mechanisms via which DAPK elicits its biological action remain largely unknown. We report here that the mammalian 40S ribosomal protein S6 is a DAPK substrate. Results from immunoprecipitation experiments are consistent with endogenous DAPK being associated with endogenous S6 in rat brain. When S6 is a component of the 40S ribosomal subunit complex, DAPK selectively phosphorylates it at serine 235, one of the five sites in S6 that are phosphorylated by the S6 kinase family of proteins. The amino acid sequence flanking serine 235 matches the established pattern for DAPK peptide and protein substrates. Kinetic analyses using purified 40S subunits revealed a K(m) value of 9 microM, consistent with S6 being a potential physiological substrate of DAPK. This enzyme-substrate relationship has functional significance. DAPK suppresses translation in rabbit reticulocyte lysate, and treatment of neuroblastoma cells with a stimulator of DAPK reduces protein synthesis. In both cases, suppression of translation correlates with increased phosphorylation of S6 at serine 235. These results demonstrate that DAPK is a S6 kinase and provide evidence for a novel role of DAPK in the regulation of translation.     

Mechanism of flavin reduction in class 2 dihydroorotate dehydrogenases

Dihydroorotate dehydrogenases (DHODs) oxidize dihydroorotate (DHO) to orotate using the FMN prosthetic group to abstract a hydride equivalent from C6 and a protein residue (Ser for Class 2 DHODs) to deprotonate C5. The fundamental question of whether the scission of the two DHO C-H bonds is concerted or stepwise was addressed for two Class 2 enzymes, those from Escherichia coli and Homo sapiens, by determining kinetic isotope effects on flavin reduction in anaerobic stopped-flow experiments. Isotope effects were determined for the E. coli enzyme at two pH values below a previously reported pKa controlling reduction [Palfey, B. A., Bj?rnberg, O., and Jensen K. F. (2001) Biochemistry 40, 4381-4390] and were about 3-fold for DHO labeled at the 5-position, about 4-fold for DHO labeled at the 6-position, and about 6-7-fold for DHO labeled at both the 5- and 6-positions. These isotope effects are consistent with either a stepwise oxidation of DHO or a concerted mechanism with significant quantum mechanical tunneling. At a pH value above the pKa controlling reduction, no isotope effect was observed in E. coli DHOD for DHO deuterated at the 5-position (the proton donor in the reaction). This is consistent with a stepwise reaction; above the (kinetic) pKa, the deprotonation of C5 is fast enough that it does not contribute to the observed rate constant and, therefore, is not isotopically sensitive. All available information points to Ser acting as a component in a proton relay network which allows its transient deprotonation. The H. sapiens DHOD also appears to have a pKa near 9.4 controlling reduction, similar to that previously reported for the E. coli enzyme. Similar KIEs were obtained with the H. sapiens enzyme at a pH value below the pKa.     

Identification of a novel, highly variable amino-terminal amino acid sequence element in the nuclear intermediate filament protein lamin B(2) from higher vertebrates

By comparing newly available cDNA sequences of the human intermediate filament protein lamin B(2) with published sequences, we have identified an additional translation initiation codon 60 nucleotides upstream of the previously assumed translation start. In addition, corresponding sequences were identified in the chimpanzee, mouse, rat and bovine genes and cDNAs, respectively. Therefore, we generated antibodies against these potential 20 new amino acids of the human sequence. By immunoblot analysis and immunofluorescence microscopy we show that human lamin B(2) is indeed synthesized as a longer version than previously reported, because it contains these additional 20 amino acids. Notably, the sequence homology to mouse, rat and bovine lamin B(2) is significantly lower in this segment than in that between the second methionine codon and the start of the alpha-helical rod indicating that the tip of the "head" is engaged in more species-specific functions. Forced expression of the GFP-tagged authentic "long" and the 20 amino acid shorter version of lamin B(2) in human cultured SW-13 cells demonstrated that both the longer and the shorter version are properly integrated into the nuclear lamina, although the shorter version exhibited a tendency to disturb envelope architecture at higher expression levels.     

Comparative modeling and analysis of microfluidic and conventional DNA microarrays

A theoretical analysis was developed to predict molecular hybridization rates for microarrays where samples flow through microfluidic channels and for conventional microarrays where samples remain stationary during hybridization. The theory was validated by using a multiplexed microfluidic microarray where eight samples were hybridized simultaneously against eight probes using 60-mer DNA strands. Mass transfer coefficients ranged over three orders of magnitude where either kinetic reaction rates or molecular diffusion rates controlled overall hybridization rates. Probes were printed using microfluidic channels and also conventional spotting techniques. Consistent with the theoretical model, the microfluidic microarray demonstrated the ability to print DNA probes in less than 1 min and to detect 10-pM target concentrations with hybridization times in less than 5 min.