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.