Active site dynamics in protein molecules: a stochastic boundary molecular-dynamics approach

The stochastic boundary molecular-dynamics approach for studying chemical events in liquids is extended to proteins. A method for simulating the localized dynamics of a portion of a protein is developed and illustrated by an application to the bovine pancreatic trypsin inhibitor (BPTI). The dynamics of the aromatic ring atoms of Tyr 21 of BPTI are targeted as the “active site,” and the results from several different localized models are compared with those from a simulation of the entire molecule. The most consistent results are obtained with a model that consists of a buffer region composed of harmonically constrained Langevin atoms surrounding the active site region whose atoms are treated by molecular dynamics. Possible applications of the model are discussed.     

A probability-based approach for high-throughput protein phosphorylation analysis and site localization

Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.     

Search by proteins for their DNA target site: 1. The effect of DNA conformation on protein sliding

The recognition of DNA-binding proteins (DBPs) to their specific site often precedes by a search technique in which proteins slide, hop along the DNA contour or perform inter-segment transfer and 3D diffusion to dissociate and re-associate to distant DNA sites. In this study, we demonstrated that the strength and nature of the non-specific electrostatic interactions, which govern the search dynamics of DBPs, are strongly correlated with the conformation of the DNA. We tuned two structural parameters, namely curvature and the extent of helical twisting in circular DNA. These two factors are mutually independent of each other and can modulate the electrostatic potential through changing the geometry of the circular DNA conformation. The search dynamics for DBPs on circular DNA is therefore markedly different compared with linear B-DNA. Our results suggest that, for a given DBP, the rotation-coupled sliding dynamics is precluded in highly curved DNA (as well as for over-twisted DNA) because of the large electrostatic energy barrier between the inside and outside of the DNA molecule. Under such circumstances, proteins prefer to hop in order to explore interior DNA sites. The change in the balance between sliding and hopping propensities as a function of DNA curvature or twisting may result in different search efficiency and speed.     

A protein target site in an early replicated human DNA sequence: a highly conserved binding motif

We have previously reported that a human nuclear factor, probably corresponding to the USF/MLTF protein [1,2], is able to bind specifically to a DNA sequence present in DNA replicated at the onset of S-phase [3]. Here we demonstrate that the same factor binds also to several other similar sequences, present in eukaryotic and viral genomes. Mutations or methylation in a CpG dinucleotide, central in the palindromic binding site, completely abolish binding. Furthermore, we present evidence for the existence of at least two other nuclear proteins in human cells with the same DNA binding specificity. The data presented suggest a strong evolutionary conservation, among distantly related organisms, of the binding motif, which is probably the target of a number of nuclear factors that share the same DNA binding specificity albeit in the context of different functions.     

Mapping DNA by stochastic relaxation: a new approach to fragment sizes

Instead of the traditional manipulations with given fixed fragmentlengths in the restriction map construction a method of varyingthe lengths is proposed and realized under the simulated annealingalgorithm scheme. The described approach has no upper limiton the number of fragments mapped with even ordinary hardware.A program has been derived from the algorithm combined withthe least–squares refinement procedure for both linearand circular maps. The algorithm's abilily to pick up missedmaps is illustrated and the problem of reducing the number ofsolutions is discussed. Received on January 8, 1990; accepted on January 9, 1990     

Retrotransposon target site selection by imitation of a cellular protein

Mobile elements rely on cellular processes to replicate, and therefore, mobile element proteins frequently interact with a variety of cellular factors. The integrase (IN) encoded by the retrotransposon Ty5 interacts with the heterochromatin protein Sir4, and this interaction determines Ty5's preference to integrate into heterochromatin. We explored the hypothesis that Ty5's targeting mechanism arose by mimicking an interaction between Sir4 and another cellular protein(s). Mutational analyses defined the requirements for the IN-Sir4 interaction, providing criteria to screen for cellular analogues. Esc1, a protein associated with the inner nuclear membrane, interacted with the same domain of Sir4 as IN, and 75% of mutations that disrupted IN-Sir4 interactions also abrogated Esc1-Sir4 interactions. A small motif critical for recognizing Sir4 was identified in Esc1. The functional equivalency of this motif and the Sir4-interacting domain of IN was demonstrated by swapping these motifs and showing that the chimeric IN and Esc1 proteins effectively target integration and partition DNA, respectively. We conclude that Ty5 targets integration by imitating the Esc1-Sir4 interaction and suggest molecular mimicry as a general mechanism that enables mobile elements to interface with cellular processes.     

Kinetics and thermodynamics of protein adsorption: a generalized molecular theoretical approach

The thermodynamics and kinetics of protein adsorption are studied using a molecular theoretical approach. The cases studied include competitive adsorption from mixtures and the effect of conformational changes upon adsorption. The kinetic theory is based on a generalized diffusion equation in which the driving force for motion is the gradient of chemical potentials of the proteins. The time-dependent chemical potentials, as well as the equilibrium behavior of the system, are obtained using a molecular mean-field theory. The theory provides, within the same theoretical formulation, the diffusion and the kinetic (activated) controlled regimes. By separation of ideal and nonideal contributions to the chemical potential, the equation of motion shows a purely diffusive part and the motion of the particles in the potential of mean force resulting from the intermolecular interactions. The theory enables the calculation of the time-dependent surface coverage of proteins, the dynamic surface tension, and the structure of the adsorbed layer in contact with the approaching proteins. For the case of competitive adsorption from a solution containing a mixture of large and small proteins, a variety of different adsorption patterns are observed depending upon the bulk composition, the strength of the interaction between the particles, and the surface and size of the proteins. It is found that the experimentally observed Vroman sequence is predicted in the case that the bulk solution is at a composition with an excess of the small protein, and that the interaction between the large protein and the surface is much larger than that of the smaller protein. The effect of surface conformational changes of the adsorbed proteins in the time-dependent adsorption is studied in detail. The theory predicts regimes of constant density and dynamic surface tension that are long lived but are only intermediates before the final approach to equilibrium. The implications of the findings to the interpretation of experimental observations is discussed.     

Studies of bacteriophage P2 DNA replication: localization of the cleavage site of the A protein.

Bacteriophage P2 replicates via a modified rolling circle-type of mechanism, where the P2 A protein acts as an initiator of the replication by inducing a single-stranded cut at the origin of replication (ori). The exact location of the cut induced by the A protein in vivo is determined in this report by: (i) restriction analysis; (ii) DNA sequence analysis; and (iii) primer extensions. It is located 89.2% from the left end of the P2 genome, which is within the coding part of the A gene, in a region devoid of secondary structures. The A gene has been cloned into an expression vector, and the A protein has been purified. The purified A protein does not bind to double-stranded ori containing DNA, but it cleaves single-stranded ori containing DNA, which indicates that a special DNA structure and/or protein is required to make the ori accessible for the A protein.     

Triplex mediated delivery of a platinum complex to a specific DNA target site

Tethering an ethylene diamine linker to the 5' terminus of an oligothymidine sequence provides a site for complexation with K(2)PtCl(4). Due to the low reactivity of dT toward a platinum source, we chose dT(8) and dT(15) as our initial synthetic targets for platination. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. The dT(8) sequence is not sufficiently long to facilitate triplex formation and Pt-cross-linking, whereas with a dT(15) sequence cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand, or of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA and the orientational control arising from triplex formation is sufficient to alter the binding preferences of platinum. Conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.     

Requirement of the FATC domain of protein kinase Tel1 for localization to DNA ends and target protein recognition

Two large phosphatidylinositol 3-kinase–related protein kinases (PIKKs), ATM and ATR, play a central role in the DNA damage response pathway. PIKKs contain a highly conserved extreme C-terminus called the FRAP-ATM-TRRAP-C-terminal (FATC) domain. In budding yeast, ATM and ATR correspond to Tel1 and Mec1, respectively. In this study, we characterized functions of the FATC domain of Tel1 by introducing substitution or truncation mutations. One substitution mutation, termed tel1-21, and a truncation mutation, called tel1-ΔC, did not significantly affect the expression level. The tel1-21 mutation impaired the cellular response to DNA damage and conferred moderate telomere maintenance defect. In contrast, the tel1-ΔC mutation behaved like a null mutation, conferring defects in both DNA damage response and telomere maintenance. Tel1-21 protein localized to DNA ends as effectively as wild-type Tel1 protein, whereas Tel1-ΔC protein failed. Introduction of a hyperactive TEL1-hy mutation suppressed the tel1-21 mutation but not the tel1-ΔC mutation. In vitro analyses revealed that both Tel1-21 and Tel1-ΔC proteins undergo efficient autophosphorylation but exhibit decreased kinase activities toward the exogenous substrate protein, Rad53. Our results show that the FATC domain of Tel1 mediates localization to DNA ends and contributes to phosphorylation of target proteins.     

NAR Breakthrough Article: The casposon-encoded Cas1 protein from Aciduliprofundum boonei is a DNA integrase that generates target site duplications

Many archaea and bacteria have an adaptive immune system known as CRISPR which allows them to recognize and destroy foreign nucleic acid that they have previously encountered. Two CRISPR-associated proteins, Cas1 and Cas2, are required for the acquisition step of adaptation, in which fragments of foreign DNA are incorporated into the host CRISPR locus. Cas1 genes have also been found scattered in several archaeal and bacterial genomes, unassociated with CRISPR loci or other cas proteins. Rather, they are flanked by nearly identical inverted repeats and enclosed within direct repeats, suggesting that these genetic regions might be mobile elements (‘casposons’). To investigate this possibility, we have characterized the in vitro activities of the putative Cas1 transposase (‘casposase’) from Aciduliprofundum boonei. The purified Cas1 casposase can integrate both short oligonucleotides with inverted repeat sequences and a 2.8 kb excised mini-casposon into target DNA. Casposon integration occurs without target specificity and generates 14–15 basepair target site duplications, consistent with those found in casposon host genomes. Thus, Cas1 casposases carry out similar biochemical reactions as the CRISPR Cas1-Cas2 complex but with opposite substrate specificities: casposases integrate specific sequences into random target sites, whereas CRISPR Cas1-Cas2 integrates essentially random sequences into a specific site in the CRISPR locus.     

SdrI of Staphylococcus saprophyticus is a multifunctional protein: localization of the fibronectin-binding site

The transferability of a large plasmid that harbors a tetracycline resistance gene tet (S), to fish and human pathogens was assessed using electrotransformation and conjugation. The plasmid, originally isolated from fish intestinal Lactococcus lactis ssp. lactis KYA-7, has potent antagonistic activity against the selected recipients ( Lactococcus garvieae and Listeria monocytogenes ), preventing conjugation. Therefore the tetracycline resistance determinant was transferred via electroporation to L . garvieae . A transformant clone was used as the donor in conjugation experiments with three different L. monocytogenes strains. To our knowledge, this is the first study showing the transfer of an antibiotic resistance plasmid from fish-associated lactic bacteria to L. monocytogenes , even if the donor L. garvieae was not the original host of the tetracycline resistance but experimentally created by electroporation. These results demonstrate that the antibiotic resistance genes in the fish intestinal bacteria have the potential to spread both to fish and human pathogens, posing a risk to aquaculture and consumer safety.     

Thermodynamics of DNA target site recognition by homing endonucleases

The thermodynamic profiles of target site recognition have been surveyed for homing endonucleases from various structural families. Similar to DNA-binding proteins that recognize shorter target sites, homing endonucleases display a narrow range of binding free energies and affinities, mediated by structural interactions that balance the magnitude of enthalpic and entropic forces. While the balance of ΔH and TΔS are not strongly correlated with the overall extent of DNA bending, unfavorable ΔH_binding is associated with unstacking of individual base steps in the target site. The effects of deleterious basepair substitutions in the optimal target sites of two LAGLIDADG homing endonucleases, and the subsequent effect of redesigning one of those endonucleases to accommodate that DNA sequence change, were also measured. The substitution of base-specific hydrogen bonds in a wild-type endonuclease/DNA complex with hydrophobic van der Waals contacts in a redesigned complex reduced the ability to discriminate between sites, due to nonspecific ΔS_binding.     

Kinetics and localization of wound-induced DNA biosynthesis in potato tuber

Tuber wounding induces a cascade of biological responses that are involved in processes required to heal and protect surviving plant tissues. Little is known about the coordination of these processes, including essential wound-induced DNA synthesis, yet they play critical roles in maintaining marketability of the harvested crop and tubers cut for seed. A sensitive “Click-iT EdU Assay” employing incorporation of the thymidine analog, 5-ethynyl-2′-deoxyuridine (EdU), in conjunction with 4′,6-diamindino-2-phenylindole (DAPI) counter labeling, was employed to objectively identify and determine the time course and spatial distribution of tuber nuclei that were wound-induced to enter S-phase of the cell cycle. Both labeling procedures are rapid and sensitive in situ. Following wounding, EdU incorporation (indicating DNA synthesis) was not detectable until after 12 h, rapidly reached a maximum at about 18 h and then declined to near zero at 48 h. About 28% of the nuclei were EdU labeled at 18 h reflecting the proportion of cells in S-phase of the cell cycle. During the ∼30 h in which induced cells were progressing through S-phase, de novo DNA synthesis extended 7–8 cell layers below the wound surface. Cessation of nuclear DNA synthesis occurred about 4 d prior to completion of wound closing layer formation. Initiation of wound periderm development followed at 7 d, i.e. about 5 d after cessation of nuclear DNA biosynthesis; at this time the phellogen developed and meristematic activity was detected via the production of new phellem cells. Collectively, these results provide new insight into the coordination of wound-induced nucleic acid synthesis with associated tuber wound-healing processes.     

Optimality of collective choices: a stochastic approach

Amplifying communication is a characteristic of group-living animals. This study is concerned with food recruitment by chemical means, known to be associated with foraging in most ant colonies but also with defence or nest moving. A stochastic approach of collective choices made by ants faced with different sources is developed to account for the fluctuations inherent to the recruitment process. It has been established that ants are able to optimize their foraging by selecting the most rewarding source. Our results not only confirm that selection is the result of a trail modulation according to food quality but also show the existence of an optimal quantity of laid pheromone for which the selection of a source is at the maximum, whatever the difference between the two sources might be. In terms of colony size, large colonies more easily focus their activity on one source. Moreover, the selection of the rich source is more efficient if many individuals lay small quantities of pheromone, instead of a small group of individuals laying a higher trail amount. These properties due to the stochasticity of the recruitment process can be extended to other social phenomena in which competition between different sources of information occurs.     

Spreading dynamics on complex networks: a general stochastic approach

Dynamics on networks is considered from the perspective of Markov stochastic processes. We partially describe the state of the system through network motifs and infer any missing data using the available information. This versatile approach is especially well adapted for modelling spreading processes and/or population dynamics. In particular, the generality of our framework and the fact that its assumptions are explicitly stated suggests that it could be used as a common ground for comparing existing epidemics models too complex for direct comparison, such as agent-based computer simulations. We provide many examples for the special cases of susceptible-infectious-susceptible and susceptible-infectious-removed dynamics (e.g., epidemics propagation) and we observe multiple situations where accurate results may be obtained at low computational cost. Our perspective reveals a subtle balance between the complex requirements of a realistic model and its basic assumptions.     

DNA affects the composition of lipoplex protein corona: a proteomics approach

The distribution of drug delivery systems into the body is affected by plasma proteins adsorbed onto their surface. Furthermore, an exact understanding of the structure and morphology of drug carriers is fundamental to understand their role as gene delivery systems. In this work, the adsorption of human plasma proteins bound to cationic liposomes and to their relative DNA lipoplexes was compared. A shotgun proteomics approach based on HPLC coupled to high resolution MS was used for an efficient identification of proteins adsorbed onto liposome and lipoplex surfaces. The distinct pattern of proteins adsorbed helps to better understand the DNA compaction process. The experimental evidence leads us to hypothesize that polyanionic DNA is associated to the lipoplex surface and can interact with basic plasma proteins. Such a finding is in agreement with recent results showing that lipoplexes are multilamellar DNA/lipid domains partially decorated with DNA at their surface. Proteomics experiments showed that the lipoplex corona is rich of biologically relevant proteins such as fibronectin, histones and complement proteins. Our results provide novel insights to understand how lipoplexes activate the immune system and why they are rapidly cleared from the blood stream. The differences in the protein adsorption data detected in the presented experiments could be the basis for the establishment of a correlation between protein adsorption pattern and in vivo fate of intravenously administered nanoparticles and will require some consideration in the future.