Hydration Changes in the Association of Hoechst 33258 with DNA

Kiser, John R.; Monk, R. W.; Smalls, Rondey L.; Petty, Jeffrey T.

doi:10.1021/bi051769x

Cited by 49 publications

(74 citation statements)

References 86 publications

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“…To determine whether the previously observed effect of sucrose on DHF binding (10) arises solely from perturbations on water activity, osmolytes with different characteristics were used. For example, addition of glycine betaine or sucrose can both affect water activity, yet these molecules either increase or decrease the dielectric constant of the solution, respectively (30,35,36). If both compounds show similar results, then effects on the dielectric constant are not involved.…”

Section: Role Of Water In K Cat /K M(dhf)mentioning

confidence: 99%

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Chopra

Dooling

Horner

et al. 2008

Journal of Biological Chemistry

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R67 dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. This enzyme is a homotetramer possessing 222 symmetry, and a single active site pore traverses the length of the protein. A promiscuous binding surface can accommodate either DHF or NADPH, thus two nonproductive complexes can form (2NADPH or 2DHF) as well as a productive complex (NADPH⅐DHF). The role of water in binding was monitored using a number of different osmolytes. From isothermal titration calorimetry (ITC) studies, binding of NADPH is accompanied by the net release of 38 water molecules. In contrast, from both steady state kinetics and ITC studies, binding of DHF is accompanied by the net uptake of water. Although different osmolytes have similar effects on NADPH binding, variable results are observed when DHF binding is probed. Sensitivity to water activity can also be probed by an in vivo selection using the antibacterial drug, trimethoprim, where the water content of the media is decreased by increasing concentrations of sorbitol. The ability of wild type and mutant clones of R67 DHFR to allow host Escherichia coli to grow in the presence of trimethoprim plus added sorbitol parallels the catalytic efficiency of the DHFR clones, indicating water content strongly correlates with the in vivo function of R67 DHFR.R67 dihydrofolate reductase, a type II DHFR, 2 catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. The gene for this enzyme is carried by an R-plasmid, and its presence confers resistance to the antibiotic drug, trimethoprim (TMP). Trimethoprim is a competitive inhibitor of chromosomal DHFR with a picomolar K i (1). Although R67 DHFR is not a good catalyst, it is not inhibited by TMP very well, and thus it allows cell growth in the presence of this antibacterial drug (2, 3). R67 DHFR shares no homology in sequence or structure with chromosomal DHFR and has been proposed to be a good model of a primitive enzyme (4).R67 DHFR is a homotetramer with a single active site pore; the overall structure possesses 222 symmetry as seen in Fig. 1 (5). The symmetry of the active site results in overlapping binding sites for substrate, DHF, and cofactor, NADPH. This can be observed as R67 DHFR binds a total of two ligands as follows: either two NADPH molecules or two folate/DHF molecules or one NADPH plus one folate/DHF molecule (6). The first two complexes are dead-end (binary) complexes, whereas the third is the productive ternary complex. Because of the 222 symmetry, binding to either ligand is unlikely to be optimal.The active site pore of R67 DHFR is unusual in its hourglass shape as well as its large size (2938 Å 3 for apoR67 DHFR with hydrogens added, calculated by CASTp (4, 7)). Because of this large volume, DHF and NADPH cannot occupy all the space in the pore and must use water to mediate some contacts with the protein.How do substrate and cofactor bind to R67 DHFR? From NMR, crystallography, and docking studies, the pteridine ring of DHF/fo...

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Section: Role Of Water In K Cat /K M(dhf)mentioning

confidence: 99%

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Chopra

Dooling

Horner

et al. 2008

Journal of Biological Chemistry

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“…4,5,7) Binding of Hoechst 33258 increases the stability of CtDNA in a concentration dependent manner. 43,44) In the presence of these osmolytes interesting observation were made in respect of lowering of T m .…”

Section: Discussionmentioning

confidence: 99%

“…1) Interaction of Hoechst 33258 has been extensively studied using oligonucleotides. [2][3][4][5] Different aspects of this interaction viz., physicochemical, thermodynamics, NMR, X-ray crystallography, in silico simulation are now well established. 3,[6][7][8][9][10][11] From the classical observation on the ability of Hoechst 33258 binding in the AT rich region of minor groove of DNA.…”

Section: Regular Articlementioning

confidence: 99%

“…3,[26][27][28][29] Most of the studies and hence our understandings are still based on oligonucleotides. 5,27,30) Water molecules located close to the binding surfaces are more structured than those of bulk solvent. These locally structured water molecules are retained or released during a binding process with different thermodynamic consequences.…”

Section: Regular Articlementioning

confidence: 99%

“…Osmotic stress method has been employed for studying the hydration changes for minor groove binder as well as for intercalators. 5,26,27,[36][37][38] In living system, small molecules interact with DNA in a very crowded environment. The presence of co-solutes in the in-vivo conditions affects the interaction process therefore it is important to study the interaction of Hoechst 33258 with genomic DNA in a very complex environment.…”

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confidence: 99%

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Osmolyte Changes the Binding Affinity and Mode of Interaction of Minor Groove Binder Hoechst 33258 with Calf Thymus DNA

Anuradha

Alam

Chaudhury

2010

CHEMICAL & PHARMACEUTICAL BULLETIN

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The bis-benzimidazole molecule Hoechst 33258 ( Fig. 1) is an important DNA minor groove binding ligand.1) Interaction of Hoechst 33258 has been extensively studied using oligonucleotides.2-5) Different aspects of this interaction viz., physicochemical, thermodynamics, NMR, X-ray crystallography, in silico simulation are now well established. 3,[6][7][8][9][10][11] From the classical observation on the ability of Hoechst 33258 binding in the AT rich region of minor groove of DNA. Synthetic chemists have designed various DNA minor groove binders for possible applications such as anticancer, antiviral, and other diseases. 12,13) Studies at cellular level have demonstrated the desired biological effects.14-16) New applications are emerging from studies related to radiosensitization and radioprotection in relation to modification of radiation response by Hoechst derivatives. [17][18][19][20][21] Attempts have also been made to correlate these observations with different Hoechst analogues.Physicochemical and thermodynamic studies have revealed important details on the mechanism of Hoechst 33258-DNA interaction. 12,[22][23][24] Role of water surrounding DNA has emerged as an important entity in the interaction processes. 12,22,25) The network of newly formed hydrogen bonds between the ligand and DNA replaces previously existing direct solute-solvent (water) hydrogen bonds in the minor groove. Hydration changes strongly influences the thermodynamics of ligand-DNA interaction. The change in hydration after the complex formation appeared to be governed by the structure of the ligand. 3,[26][27][28][29] Most of the studies and hence our understandings are still based on oligonucleotides. 5,27,30) Water molecules located close to the binding surfaces are more structured than those of bulk solvent. These locally structured water molecules are retained or released during a binding process with different thermodynamic consequences. Various techniques are available to study hydration changes accompanying ligand-DNA interaction 31,32) and osmotic stress approach has gained importance due to its ability to specifically alter water activity of the surrounding solution; a fact that has been harnessed to measure forces between molecules. 31,32) It is relatively a simple technique to study the hydration changes upon ligand-DNA interaction and gives stoichiometric amount of water release or uptake upon biomolecular complexation.27) The principle of osmotic stress method is based on Gibbs-Duhem equation. 34,35) The neutral solutes are added directly to the solution containing the ligand and macromolecule causes changes in the water activity of the solution. Osmotic stress method has been employed for studying the hydration changes for minor groove binder as well as for intercalators. 5,26,27,[36][37][38] In living system, small molecules interact with DNA in a very crowded environment. The presence of co-solutes in the in-vivo conditions affects the interaction process therefore it is important to study the interaction of Hoechst 33258...

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