We show that the luminescence from CdSe quantum dot monolayers can be strongly influenced by the interaction of water molecules adsorbed on the surface. Light-induced alterations in the surface states following adsorption of water, results in quasi-reversible luminescence changes in the quantum dot. The excitonic QY increases by a factor of 20 during the first 200 s of illumination in air (post vacuum) and then steadily decreases to a level 6 times that of the vacuum reference after 5000 s. The exciton emission exhibits an exponential blue shift of nearly 16 nm (60 meV) over 1 h of illumination. During this time, the line width decreases by 10% during the first 100 s and then slowly increases to 96% of the vacuum reference line width after 5000 s. Our model suggests that water molecules adsorbed on the surface of the quantum dot act to passivate surface traps, which results in increased luminescence, similar to an effect well-known for bulk CdSe surfaces. In addition, adsorbed water molecules act to oxidize the surface of the quantum dot, which results in the blue shift of the exciton emission and eventually introduces new surface defects that lower the luminescence. It is the competition between these two processes that is responsible for the complex kinetics of the luminescence QY.

Molecular dynamics (MD) simulations of HhaI DNA methyltransferase and statistical coupling analysis (SCA) data on the DNA cytosine methyltransferase family were combined to identify residues that are coupled by coevolution and motion. The highest ranking correlated pairs from the data matrix product (SCA⅐MD) are colocalized and form stabilizing interactions; the anticorrelated pairs are separated on average by 30 Å and form a clear focal point centered near the active site. We suggest that these distal anticorrelated pairs are involved in mediating active-site compressions that may be important for catalysis. Mutants that disrupt the implicated interactions support the validity of our combined SCA⅐MD approach.anticorrelated motion ͉ correlated motion ͉ M.HhaI ͉ statistical coupling analysis T he proposal that protein dynamics contributes significantly to enzyme catalysis is intriguing (1-4) yet is supported by limited experimental evidence. Previous studies have shown that correlated and anticorrelated motions within an enzyme's active site enhance the reaction rate by various mechanisms that increase the relative amounts of reactive orientations (5). These active-site fluctuations are proposed to result from motions involving distal structural elements and interconnecting networks (1-4). This hypothesis is indirectly supported by emerging molecular dynamics (MD) (1, 2), NMR (6, 7), and hybrid approaches (8-12). The MD studies, although difficult to verify experimentally, have provided highly suggestive results relating dynamics to catalysis. Ultimately, the quantitative contribution to catalysis of various dynamic mechanisms requires direct experimental testing. We combined MD simulations and a coevolution analysis [statistical coupling analysis (SCA); ref. 13] to identify residues that are coupled by coevolution and motion.Although MD simulations reveal active-site correlated and anticorrelated motions, the identity and role of specific structural elements outside the active site in mediating such motions is difficult to assign. For example, MD cross-correlation analyses are dominated by anticorrelated motions occurring between the most distal regions of protein, often residing in distinct domains (5). Although MD simulations implicate regions of allowed motion, the identity of single amino acids that facilitate these motions is not forthcoming and hence difficult for protein engineers to test. SCA identifies the functional coupling of specific residue pairs that in many cases are distal in the three-dimensional structure. The coupling of such residues leads to their coevolution and is revealed by the statistical analysis of hundreds of related sequences; this approach recently was validated by NMR and protein engineering studies (13-16). These applications of SCA have been focused on protein-ligand interactions, and here we apply SCA toward protein dynamics and catalysis.M.HhaI is one of many S-adenosylmethionine (AdoMet)-dependent DNA-modifying enzymes found in bacteria, plants, and animals (17). These enzym...

The characterization of conformational changes that drive induced-fit mechanisms and their quantitative importance to enzyme specificity are essential for a full understanding of enzyme function. Here, we report on M.HhaI, a sequence-specific DNA cytosine C 5 methyltransferase that reorganizes a flexible loop (residues 80 -100) upon binding cognate DNA as part of an induced-fit mechanism. To directly observe this ϳ26 Å conformational rearrangement and provide a basis for understanding its importance to specificity, we replaced loop residues Lys-91 and Glu-94 with tryptophans. The double mutants W41F/K91W and W41F/E94W are relatively unperturbed in kinetic and thermodynamic properties. Ligand-induced changes in protein conformation can contribute to enzyme catalysis, regulation of function, and substrate specificity. Induced-fit mechanisms involve conformational rearrangements of an enzyme to facilitate or enhance correct substrate binding and can provide improved specificity (1-3). First introduced in 1958 (4), induced-fit mechanisms contribute to diverse biological processes (5), including tRNA binding in ribosomes (6), DNA-modifying enzymes (7-9), kinases (10, 11), RNA folding (12), DNA binding specificity (13,14), polymerases (15, 16), and many others. However, direct evidence for an induced-fit mechanism through the observation of real-time protein conformational rearrangements coupled to specificity have been quantitated for a small number of enzymes (17)(18)(19)(20).Enzymes that sequence-specifically modify DNA, including nucleases, repair enzymes, and methyltransferases are faced with severe challenges of substrate recognition and specificity due to the overwhelming abundance of sites that are closely related to the cognate sequence (1,3,5,21). Mechanisms posited to account for this discrimination are diverse and often require an induced-fit process as the enzyme-DNA complex moves from a nonspecific site to the cognate sequence (22, 23). The availability of high resolution structures of cognate DNAenzyme complexes for many such systems provides a detailed understanding of specific interactions leading to tight and cognate binding (24 -27). However, interactions leading to nonspecific substrate binding, which facilitate site searching, are far less characterized (1, 3, 28). Furthermore, the interconversion of conformers as the enzyme goes from nonspecific to cognate sites prior to forming the catalytically competent complex can contribute to such specificity (13, 21, 28 -31).The DNA cytosine C 5 methyltransferase M.HhaI binds DNA substrates between its two domains and the cofactor AdoMet 2 in the large domain near the active site (see Fig. 1). Inspection of two cocrystal structures of M.HhaI, one involving the cognate DNA and the cofactor product AdoHcy (3MHT.pdb) (24), the other involving nonspecific DNA and AdoHcy (2HMY.pdb) (32), suggest that the enzyme may exploit an induced-fit mechanism. Induced-fit DNA binding was first proposed for M.HhaI in 1994 when the first ternary complex with cognate DNA, cof...

We have characterized Escherichia coli DNA adenine methyltransferase, a critical regulator of bacterial virulence. Steady-state kinetics, product inhibition, and isotope exchange studies are consistent with a kinetic mechanism in which the cofactor S-adenosylmethionine binds first, followed by sequence-specific DNA binding and catalysis. The enzyme has a fast methyl transfer step followed by slower product release steps, and we directly demonstrate the competence of the enzyme cofactor complex. Methylation of adjacent GATC sites is distributive with DNA derived from a genetic element that controls the transcription of the adjacent genes. This indicates that the first methylation event is followed by enzyme release. The affinity of the enzyme for both DNA and S-adenosylmethionine was determined. Our studies provide a basis for further structural and functional analysis of this important enzyme and for the identification of inhibitors for potential therapeutic applications.Bacterial DNA methyltransferases generate N 4 -methylcytosine, C 5 -methylcytosine, and N 6 -methyladenosine in an S-adenosylmethionine-dependent reaction (1). Bacterial DNA methylation plays critical roles, including DNA repair, phage protection, gene regulation, and DNA replication, in diverse biological pathways. The majority of DNA methyltransferases form one-half of a restriction-modification system that protects the host bacteria against bacteriophage infection. Together with cognate restriction endonucleases, which generally cleave a short palindromic sequence, these restriction-modification systems provide the foundation for many recombinant DNA manipulations; the endonucleases and methyltransferases have provided many structural and mechanistic insights into the process of sequence-specific DNA recognition and modification.Not all DNA methyltransferases have an endonuclease partner or at least one which is known. Thus, DNA adenine methyltransferase (DAM, 1 methylates the adenine in GATC) in ␥-proteobacteria (2, 3), and the cell cycle-regulated methyltransferase (CcrM, methylates the adenine in GANTC) in ␣-proteobacteria (3, 4) are involved in post-replicative mismatch repair, DNA replication timing, cell cycle regulation, and the control of gene expression. DAM and CcrM have been identified as new targets for antibiotic development (5) because some pathogenic bacteria are either avirulent or not viable when the corresponding genes are removed. DNA adenine methylation regulates the pili formation genes in Escherichia coli and Salmonella, providing one of the first and clearest examples of epigenetic gene regulation (2). This DNA-mediated gene regulation involves differentially methylated GATC sites, which represent a small minority of the ϳ5,000 -20,000 GATC sites found in a typical bacterial genome.E. coli DAM is a functional monomer of 278 amino acids (6). Our present understanding of how this enzyme functions is based largely on a small number of reports (6 -10). Herman and Modrich (6) first characterized the enzyme with plasmid DNA, ...

Gold nanoparticles were modified with RNA and utilized to detect specific DNA sequences and various RNA nucleases.

Enzymes that modify DNA are faced with significant challenges in specificity for both substrate binding and catalysis. We describe how single hydrogen bonds between M.HhaI, a DNA cytosine methyltransferase, and its DNA substrate regulate the positioning of a peptide loop which is ϳ28 Å away. Stopped-flow fluorescence measurements of a tryptophan inserted into the loop provide real-time observations of conformational rearrangements. These long-range interactions that correlate with substrate binding and critically, enzyme turnover, will have broad application to enzyme specificity and drug design for this medically relevant class of enzymes.Sequence-specific modification of DNA is essential for nearly all forms of life and contributes to a myriad of biological processes including gene regulation, mismatch repair, host defense, DNA replication, and genetic imprinting. Methylation of cytosine and adenine bases is a key epigenetic process whereby phenotypic changes are inherited without altering the DNA sequence (1). The central role of the bacterial and mammalian S-adenosylmethionine (AdoMet) 2 -dependent DNA methyltransferases in virulence regulation and tumorigenesis, respectively, have led these enzymes to be validated targets for antibiotic and cancer therapies (2, 3). However, AdoMet-dependent enzymes catalyze diverse reactions, and the design of potent and selective DNA methyltransferase inhibitors is particularly challenging (4, 5). The design of drugs that bind outside the active site is a particularly attractive means of inhibition for enzymes with common cofactors like AdoMet because off-target inhibition often leads to toxicity (6). Unfortunately, robust methods to identify and characterize such critical binding sites distal from the active site have not been developed.DNA methyltransferases bind to a particular DNA sequence, stabilize the target base into an extrahelical position within the enzyme active site, and transfer the methyl moiety from AdoMet to the DNA (7). During this process, dramatic changes in the DNA structure such as bending, base flipping, or the intercalation of residues into the recognition sequence are often accompanied by large scale protein rearrangements (8).Here we characterized a specific conformational rearrangement of M.HhaI, a model DNA cytosine C 5 methyltransferase with a cognate recognition sequence of 5Ј-GCGC-3Ј. Many structures of M.HhaI are available at high resolution including an ensemble of complexes with either cognate or nonspecific DNA (9, 10). Reorganization of an essential catalytic loop (residues 80 -100) is regulated by sequence-specific protein-DNA interactions that occur ϳ28 Å away from the catalytic loop (Fig. 1). Our work quantifies the importance of such distal communication in sequence-specific DNA modification and provides plausible structural mechanisms.DNA-dependent positioning of the catalytic loop in M.HhaI was first observed crystallographically; cognate DNA stabilizes the loop-closed conformer, while nonspecific DNA leaves the loop in the open c...

Electrostatic assembly of cationic nanoparticles onto the negatively charged backbone of double-stranded DNA has been shown to produce one-dimensional chains with potential use as nanoelectronic components. In this paper, micron long DNA templates stretched on aminosilane- and hexamethyldisilazane-modified silicon surfaces are used to assemble 3.5 nm gold nanoparticles passivated with cationic thiocholine. Atomic force microscopy is used to analyze the density and defects along the approximately 5 nm high structures, with comparison between positively charged and neutral surfaces. Low background adsorption of nanoparticles is facilitated by both these surface chemistries, while the neutral surface yields a more densely packed assembly.

The molecular basis of enzymatic catalysis is of broad interest, with implications for biocatalyst design and drug development. The abundance of detailed three-dimensional structures and investigational methods provides newly addressable aspects of enzymatic function. We are interested in the importance of protein motion, and particularly correlated motions, to catalysis. The underlying premise is that protein-solvent interactions are converted into peptide motions, resulting in the transient stabilization of active site elements with preferred reactivities (1, 2).Recent studies (1) have provided highly suggestive evidence for this concept. Molecular dynamics investigations of dihydrofolate reductase demonstrate that strong coupled motions in the reactive complex disappear in the product complexes, indicating that these motions may be linked to catalysis. Mutants that alter the kinetics of particular catalytic steps are concentrated within segments of the protein structure shown to participate in highly correlated motions (1). Solid state NMR and solution NMR relaxation studies have measured substrate and protein dynamics that are matched to the turnover time of the respective enzymes (3). Studies of hydrogen and electron tunneling during enzyme catalysis provide further evidence for the importance of protein dynamics to catalytic events at the active site (4, 5).Molecular dynamic simulations of catechol O-methyltransferase and M.HhaI 1 DNA methyltransferase provided initial evidence for correlated motions within the active sites of these enzymes (6, 7). We sought to test the importance to catalysis of motions made by specific distal residues (His 127 -Thr 132 ) in facilitating active site chemistries by altering the position and orientation of critical residues such as Val 121 . Alanine scan point mutagenesis and kinetic characterization of individual steps in the catalytic cycle were used to probe the effects of such mutations and provide insights into the roles of correlated motions. M.HhaI provides an excellent structurally and functionally tractable enzyme to study various aspects of catalysis, including base flipping and the importance of motions to catalysis. M.HhaI, from Haemophilus haemolyticus, is an AdoMetdependent C 5 -cytosine methyltransferase that methylates the central cytosine (C) in the recognition sequence 5Ј-GCGC-3Ј after stabilizing the target base in an extrahelical position. Many M.HhaI crystal structures provide structural insights into the mechanisms of DNA methylation and base flipping (8). Functional analysis of the WT M.HhaI has been extensive (9 -12), including K D DNA determination for a variety of DNA substrates (13,14). Many structural components of the M.HhaI mechanism have been examined by mutagenesis including Gln 237 , which positions itself into the DNA helix and interacts with the lone guanine (15), and Cys 81 , which forms a covalent bond to the target cytosine (16). Other mutational studies have examined protein-phosphate interactions (17) and conserved residues within the...