Functional analysis of conserved motifs in EcoP15I DNA methyltransferase

EcoP15I DNA methyltransferase recognizes the sequence 5'-CAGCAG-3' and transfers a methyl group to N-6 of the second adenine residue in the recognition sequence. All N-6 adenine methyltransferases contain two highly conserved sequences, FxGxG (motif I), postulated to form part of the S-adenosyl-L-methionine binding site and (D/N/S)PP(Y/F) (motif IV) involved in catalysis. We have altered the second glycine residue in motif I to arginine and serine, and substituted tyrosine in motif IV with tryptophan in EcoP15I DNA methyltransferase, using site-directed mutagenesis. The mutant enzymes were overexpressed, purified and characterized by biochemical methods. The mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered. Although the mutation in motif IV resulted in loss of enzyme activity, we observed enhanced crosslinking of S-adenosyl-L-methionine and DNA. This implies that DNA and AdoMet binding sites are close to motif IV. Taken together, these results reinforce the importance of motif I in AdoMet binding and motif IV in catalysis. Additionally, limited proteolysis and UV crosslinking experiments with EcoP15I DNA methyltransferase imply that DNA binds in a cleft formed by two domains in the protein. Methylation protection analysis provides evidence for the fact that EcoP15I DNA MTase makes contacts in the major groove of its substrate DNA. Interestingly, hypermethylation of the guanine residue next to the target adenine residue indicates that the protein probably flips out the target adenine residue. (C) 1996 Academic Press Limited

BER Analysis of Uplink OFDMA in the Presence of Carrier Frequency and Timing Offsets on Rician Fading Channels

In uplink orthogonal frequency division multiple access (OFDMA), carrier frequency offsets (CFO) and/or timing offsets (TO) of other users with respect to a desired user can cause significant multiuser interference (MUI). In this paper, we derive an analytical bit error rate (BER) expression that quantify the degradation in BER due to the combined effect of both CFOs and TOs in uplink OFDMA on Rician fading channels. Such an analytical BER derivation for uplink OFDMA with CFOs and TOs on Rician fading channels has not been reported so far. For the case of non-zero CFOs/TOs, we obtain an approximate BER expression involving a single integral. Analytical and simulation BER results are shown to match very well.

BER Analysis of Uplink OFDMA in the Presence of Carrier Frequency and Timing Offsets on Rician Fading Channels

In orthogonal frequency-division multiple access (OFDMA) on the uplink, the carrier frequency offsets (CFOs) and/or timing offsets (TOs) of other users with respect to a desired user can cause multiuser interference (MUI). Analytically evaluating the effect of these CFO/TO-induced MUI on the bit error rate (BER) performance is of interest. In this paper, we analyze the BER performance of uplink OFDMA in the presence of CFOs and TOs on Rician fading channels. A multicluster multipath channel model that is typical in indoor/ultrawideband and underwater acoustic channels is considered. Analytical BER expressions that quantify the degradation in BER due to the combined effect of both CFOs and TOs in uplink OFDMA with M-state quadrature amplitude modulation (QAM) are derived. Analytical and simulation BER results are shown to match very well. The derived BER expressions are shown to accurately quantify the performance degradation due to nonzero CFOs and TOs, which can serve as a useful tool in OFDMA system design.

Functional Analysis of DNA Replication Fork Reversal Catalyzed by Mycobacterium tuberculosis RuvAB Proteins

Initially discovered in Escherichia coli, RuvAB proteins are ubiquitous in bacteria and play a dual role as molecular motor proteins responsible for branch migration of the Holliday junction(s) and reversal of stalled replication forks. Despite mounting genetic evidence for a crucial role of RuvA and RuvB proteins in reversal of stalled replication forks, the mechanistic aspects of this process are still not fully understood. Here, we elucidate the ability of Mycobacterium tuberculosis RuvAB (MtRuvAB) complex to catalyze the reversal of replication forks using a range of DNA replication fork substrates. Our studies show that MtRuvAB, unlike E. coli RuvAB, is able to drive replication fork reversal via the formation of Holliday junction intermediates, suggesting that RuvAB-catalyzed fork reversal involves concerted unwinding and annealing of nascent leading and lagging strands. We also demonstrate the reversal of replication forks carrying hemi-replicated DNA, indicating that MtRuvAB complex-catalyzed fork reversal is independent of symmetry at the fork junction. The fork reversal reaction catalyzed by MtRuvAB is coupled to ATP hydrolysis, is processive, and culminates in the formation of an extended reverse DNA arm. Notably, we found that sequence heterology failed to impede the fork reversal activity of MtRuvAB. We discuss the implications of these results in the context of recognition and processing of varied types of replication fork structures by RuvAB proteins.

Functional Analysis of the Genes Encoding Diaminopropionate Ammonia Lyase in Escherichia coli and Salmonella enterica Serovar Typhimurium

Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the conversion of diaminopropionate (DAP) to pyruvate and ammonia and plays an important role in cell metabolism. We have investigated the role of the ygeX gene of Escherichia coli K-12 and its ortholog, STM1002, in Salmonella enterica serovar Typhimurium LT2, presumed to encode DAPAL, in the growth kinetics of the bacteria. While Salmonella Typhimurium LT2 could grow on DL-DAP as a sole carbon source, the wild-type E. coli K-12 strain exhibited only marginal growth on DL-DAP, suggesting that DAPAL is functional in S. Typhimurium. The expression of ygeX in E. coli was low as detected by reverse transcriptase PCR (RT-PCR), consistent with the poor growth of E. coli on DL-DAP. Strains of S. Typhimurium and E. coli with STM1002 and ygeX, respectively, deleted showed loss of growth on DL-DAP, confirming that STM1002 (ygeX) is the locus encoding DAPAL. Interestingly, the presence of DL-DAP caused a growth inhibition of the wild-type E. coli strain as well as the knockout strains of S. Typhimurium and E. coli in minimal glucose/glycerol medium. Inhibition by DL-DAP was rescued by transforming the strains with plasmids containing the STM1002 (ygeX) gene encoding DAPAL or supplementing the medium with Casamino Acids. Growth restoration studies using media lacking specific amino acid supplements suggested that growth inhibition by DL-DAP in the absence of DAPAL is associated with auxotrophy related to the inhibition of the enzymes involved in the biosynthetic pathways of pyruvate and aspartate and the amino acids derived from them.

Functional analysis of ultra high information rates conveyed by rat vibrissal primary afferents

Sensory receptors determine the type and the quantity of information available for perception. Here, we quantified and characterized the information transferred by primary afferents in the rat whisker system using neural system identification. Quantification of ``how much'' information is conveyed by primary afferents, using the direct method (DM), a classical information theoretic tool, revealed that primary afferents transfer huge amounts of information (up to 529 bits/s). Information theoretic analysis of instantaneous spike-triggered kinematic stimulus features was used to gain functional insight on ``what'' is coded by primary afferents. Amongst the kinematic variables tested-position, velocity, and acceleration-primary afferent spikes encoded velocity best. The other two variables contributed to information transfer, but only if combined with velocity. We further revealed three additional characteristics that play a role in information transfer by primary afferents. Firstly, primary afferent spikes show preference for well separated multiple stimuli (i.e., well separated sets of combinations of the three instantaneous kinematic variables). Secondly, neurons are sensitive to short strips of the stimulus trajectory (up to 10 ms pre-spike time), and thirdly, they show spike patterns (precise doublet and triplet spiking). In order to deal with these complexities, we used a flexible probabilistic neuron model fitting mixtures of Gaussians to the spike triggered stimulus distributions, which quantitatively captured the contribution of the mentioned features and allowed us to achieve a full functional analysis of the total information rate indicated by the DM. We found that instantaneous position, velocity, and acceleration explained about 50% of the total information rate. Adding a 10 ms pre-spike interval of stimulus trajectory achieved 80-90%. The final 10-20% were found to be due to non-linear coding by spike bursts.

A Genetic Analysis of the Functional Interactions within Mycobacterium tuberculosis Single-Stranded DNA Binding Protein

Single-stranded DNA binding proteins (SSBs) are vital in all organisms. SSBs of Escherichia coli (EcoSSB) and Mycobacterium tuberculosis (MtuSSB) are homotetrameric. The N-terminal domains (NTD) of these SSBs (responsible for their tetramerization and DNA binding) are structurally well defined. However, their C-terminal domains (CTD) possess undefined structures. EcoSSB NTD consists of beta 1-beta 1'-beta 2-beta 3-alpha-beta 4-beta 45(1)-beta 45(2)-beta 5 secondary structure elements. MtuSSB NTD includes an additional beta-strand (beta 6) forming a novel hook-like structure. Recently, we observed that MtuSSB complemented an E. coli Delta ssb strain. However, a chimeric SSB (m beta 4-beta 5), wherein only the terminal part of NTD (beta 4-beta 5 region possessing L-45 loop) of EcoSSB was substituted with that from MtuSSB, failed to function in E. coli in spite of its normal DNA binding and oligomerization properties. Here, we designed new chimeras by transplanting selected regions of MtuSSB into EcoSSB to understand the functional significance of the various secondary structure elements within SSB. All chimeric SSBs formed homotetramers and showed normal DNA binding. The m beta 4-beta 6 construct obtained by substitution of the region downstream of beta 5 in m beta 4-beta 5 SSB with the corresponding region (beta 6) of MtuSSB complemented the E. coli strain indicating a functional interaction between the L-45 loop and the beta 6 strand of MtuSSB.

Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance

In many organisms ``Universal Stress Proteins'' CUSPS) are induced in response to a variety of environmental stresses. Here we report the structures of two USPs, YnaF and YdaA from Salmonella typhimurium determined at 1.8 angstrom and 2.4 angstrom resolutions, respectively. YnaF consists of a single USP domain and forms a tetrameric organization stabilized by interactions mediated through chloride ions. YdaA is a larger protein consisting of two tandem USP domains. Two protomers of YdaA associate to form a structure similar to the YnaF tetramer. YdaA showed ATPase activity and an ATP binding motif G-2X-G-9X-G(S/T/N) was found in its C-terminal domain. The residues corresponding to this motif were not conserved in YnaF although YnaF could bind ATP. However, unlike YdaA, YnaF did not hydrolyse ATP in vitro. Disruption of interactions mediated through chloride ions by selected mutations converted YnaF into an ATPase. Residues that might be important for ATP hydrolysis could be identified by comparing the active sites of native and mutant structures. Only the C-terminal domain of YdaA appears to be involved in ATP hydrolysis. The structurally similar N-terminal domain was found to bind a zinc ion near the segment equivalent to the phosphate binding loop of the C-terminal domain. Mass spectrometric analysis showed that YdaA might bind a ligand of approximate molecular weight 800 daltons. Structural comparisons suggest that the ligand, probably related to an intermediate in lipid A biosynthesis, might bind at a site close to the zinc ion. Therefore, the N-terminal domain of YdaA binds zinc and might play a role in lipid metabolism. Thus, USPs appear to perform several distinct functions such as ATP hydrolysis, altering membrane properties and chloride sensing. (C) 2015 Elsevier Inc. All rights reserved.

Fatigue Laws Via Functional Equations

In routine industrial design, fatigue life estimation is largely based on S-N curves and ad hoc cycle counting algorithms used with Miner's rule for predicting life under complex loading. However, there are well known deficiencies of the conventional approach. Of the many cumulative damage rules that have been proposed, Manson's Double Linear Damage Rule (DLDR) has been the most successful. Here we follow up, through comparisons with experimental data from many sources, on a new approach to empirical fatigue life estimation (A Constructive Empirical Theory for Metal Fatigue Under Block Cyclic Loading', Proceedings of the Royal Society A, in press). The basic modeling approach is first described: it depends on enforcing mathematical consistency between predictions of simple empirical models that include indeterminate functional forms, and published fatigue data from handbooks. This consistency is enforced through setting up and (with luck) solving a functional equation with three independent variables and six unknown functions. The model, after eliminating or identifying various parameters, retains three fitted parameters; for the experimental data available, one of these may be set to zero. On comparison against data from several different sources, with two fitted parameters, we find that our model works about as well as the DLDR and much better than Miner's rule. We finally discuss some ways in which the model might be used, beyond the scope of the DLDR.

Comparative kinomics of human and chimpanzee reveal unique kinship and functional diversity generated by new domain combinations

Background: Phosphorylation by protein kinases is a common event in many cellular processes. Further, many kinases perform specialized roles and are regulated by non-kinase domains tethered to kinase domain. Perturbation in the regulation of kinases leads to malignancy. We have identified and analysed putative protein kinases encoded in the genome of chimpanzee which is a close evolutionary relative of human. Result: The shared core biology between chimpanzee and human is characterized by many orthologous protein kinases which are involved in conserved pathways. Domain architectures specific to chimp/human kinases have been observed. Chimp kinases with unique domain architectures are characterized by deletion of one or more non-kinase domains in the human kinases. Interestingly, counterparts of some of the multi-domain human kinases in chimp are characterized by identical domain architectures but with kinase-like non-kinase domain. Remarkably, out of 587 chimpanzee kinases no human orthologue with greater than 95% sequence identity could be identified for 160 kinases. Variations in chimpanzee kinases compared to human kinases are brought about also by differences in functions of domains tethered to the catalytic kinase domain. For example, the heterodimer forming PB1 domain related to the fold of ubiquitin/Ras-binding domain is seen uniquely tethered to PKC-like chimpanzee kinase. Conclusion: Though the chimpanzee and human are evolutionary very close, there are chimpanzee kinases with no close counterpart in the human suggesting differences in their functions. This analysis provides a direction for experimental analysis of human and chimpanzee protein kinases in order to enhance our understanding on their specific biological roles.

Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver

A number of studies in yeast have shown that DNA topoisomerase TI is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I. Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase rr in chromosome disjunction. As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver. The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass. Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively. The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-foe extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme. Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg2+-dependent manner. However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies. Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme. Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration. These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.

Analysis of proteins with the `hot dog' fold: Prediction of function and identification of catalytic residues of hypothetical proteins

Background: The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. Results: We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. Conclusion: The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.

Review of continuum, finite element and hybrid techniques in the analysis of stress concentrations in structures

When a uniform flow of any nature is interrupted, the readjustment of the flow results in concentrations and rare-factions, so that the peak value of the flow parameter will be higher than that which an elementary computation would suggest. When stress flow in a structure is interrupted, there are stress concentrations. These are generally localized and often large, in relation to the values indicated by simple equilibrium calculations. With the advent of the industrial revolution, dynamic and repeated loading of materials had become commonplace in engine parts and fast moving vehicles of locomotion. This led to serious fatigue failures arising from stress concentrations. Also, many metal forming processes, fabrication techniques and weak-link type safety systems benefit substantially from the intelligent use or avoidance, as appropriate, of stress concentrations. As a result, in the last 80 years, the study and and evaluation of stress concentrations has been a primary objective in the study of solid mechanics. Exact mathematical analysis of stress concentrations in finite bodies presents considerable difficulty for all but a few problems of infinite fields, concentric annuli and the like, treated under the presumption of small deformation, linear elasticity. A whole series of techniques have been developed to deal with different classes of shapes and domains, causes and sources of concentration, material behaviour, phenomenological formulation, etc. These include real and complex functions, conformal mapping, transform techniques, integral equations, finite differences and relaxation, and, more recently, the finite element methods. With the advent of large high speed computers, development of finite element concepts and a good understanding of functional analysis, it is now, in principle, possible to obtain with economy satisfactory solutions to a whole range of concentration problems by intelligently combining theory and computer application. An example is the hybridization of continuum concepts with computer based finite element formulations. This new situation also makes possible a more direct approach to the problem of design which is the primary purpose of most engineering analyses. The trend would appear to be clear: the computer will shape the theory, analysis and design.

Analysis of human chorionic gonadotropin-monoclonal antibody interaction in BIAcore

Kinetic studies of macromolecular ligand-ligate interaction have generated ample interest since the advent of plasmon resonance based instruments like BIAcore. Most of the studies reported in literature assume a simple 1 : 1 Langmuir binding and complete reversibility of the system. However we observed that in a high affinity antigen-antibody system [human chorionic gonadotropin-monoclonal antibody (hCG-mAb)] dissociation is insignificant and the sensogram data cannot be used to measure the equilibrium and kinetic parameters. At low concentrations of mAb the complete sensogram could be fitted to a single exponential. Interestingly we found that at higher mAb concentrations, the binding data did not conform to a simple bimolecular model. Instead, the data fitted a two-step model, which may be because of surface heterogeneity of affinity sites. In this paper, we report on the global fit of the sensograms. We have developed a method by which a single two-minute sensogram can be used in high affinity systems to measure the association rate constant of the reaction and the functional capacity of the ligand (hCG) immobilized on the chip. We provide a rational explanation for the discrepancies generally observed in most of the BIAcore sensograms