Femtosecond Charge Transfer Dynamics of a Modified DNA Base: 2-Aminopurine in Complexes with Nucleotides

Fiebig, Torsten; Wan, Chaozhi; Zewail, Ahmed H.

doi:10.1002/1439-7641(20020916)3:9<781::aid-cphc781>3.0.co;2-u

Cited by 109 publications

(142 citation statements)

References 38 publications

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“…Femtosecond fluorescence up-conversion procedures have been described, and we used two laser systems (21,22) for entirely independent sets of experiments. Ap is excited by a pump pulse at 320 nm.…”

Section: Methodsmentioning

confidence: 76%

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The RNA–protein complex: Direct probing of the interfacial recognition dynamics and its correlation with biological functions

Xia

Becker

Wan

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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The N protein from bacteriophage is a key regulator of transcription antitermination. It specifically recognizes a nascent mRNA stem loop termed boxB, enabling RNA polymerase to read through downstream terminators processively. The stacking interaction between Trp-18 of WT N protein and A7 of boxB RNA is crucial for efficient antitermination. Here, we report on the direct probing of the dynamics for this interfacial binding and the correlation of the dynamics with biological functions. Specifically, we examined the influence of structural changes in four peptides on the femtosecond dynamics of boxB RNA (2-aminopurine labeled in different positions), through mutations of critical residues of N peptide (residues 1-22). We then compare their in vivo (Escherichia coli) transcription antitermination activities with the dynamics. The results demonstrate that the RNA-peptide complexes adopt essentially two dynamical conformations with the time scale for interfacial interaction in the two structures being vastly different, 1 ps for the stacked structure and nanosecond for the unstacked one; only the weighted average of the two is detected in NMR by nuclear Overhauser effect experiments. Strikingly, the amplitude of the observed ultrafast dynamics depends on the identity of the amino acid residues that are one helical turn away from Trp-18 in the peptides and is correlated with the level of biological function of their respective full-length proteins.M acromolecular assemblies play critical roles in biological processes, such as expression (1), recognition (2, 3), and catalysis (4, 5). In these assemblies, the interactions are selective in forming unique structures with dynamics governing the functions. One such complex is that of RNA with proteins. It involves the bacteriophage N protein, which binds a cis-acting stem-loop RNA structure (termed boxB) in nascent mRNA coded in the phage genome, and regulates transcription elongation and termination (6-8). With other host protein factors, the complex enables RNA polymerase to read through intrinsic and Rho-dependent terminators in a processive manner (9).NMR structural studies (10-12) demonstrated that the amino terminal of the N protein (N peptide), which features an arginine-rich motif, binds to the boxB RNA hairpin as a bent ␣-helix, and this process enforces the purine-rich boxB RNA pentaloop to adopt a canonical GNRA fold (13, 14) with the fourth purine residue extruded (Fig. 1). One unique interaction featured in this RNA-peptide complex is that of the side chain of the tryptophan residue (Trp-18), which directly stacks on adenine 7, extending the RNA -stack by one residue (Fig. 1). Different peptide ligands can target boxB RNA with strong equilibrium affinities (nanomolar range for dissociation constant) similar to the WT (15), as we have shown by in vitro protein selection via mRNA display (16,17). However, examination of the energetics and structures by NMR, CD, and steady-state fluorescence on some of the selected 14͞15 mutants (Fig. 1) reveal that they have quit...

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Section: Methodsmentioning

confidence: 76%

“…In all of the experiments, the temperature of the sample quartz cell was controlled at 20°C or room temperature, and the samples were stirred. All of the data were fitted to multiexponential decay convoluted by a Gaussian response function as described (21). Transient absorption data have also been obtained and will be detailed elsewhere.…”

Section: Methodsmentioning

confidence: 99%

The RNA–protein complex: Direct probing of the interfacial recognition dynamics and its correlation with biological functions

Xia

Becker

Wan

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…It was demonstrated (17,18) that the main nonradiative pathway of the Ap in DNA is an electron transfer mechanism between Ap and neighboring bases. The decrease in emission intensity of Ap in DNA The spectra for Ap in 40% formamide, dodecamer duplex, and DNA-drug complex were multiplied by 1͞5, 10, and 10, respectively.…”

Section: Resultsmentioning

confidence: 99%

Site- and sequence-selective ultrafast hydration of DNA

Pal

Zhao

Xia

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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107

137

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Water molecules in the DNA grooves are critical for maintaining structural integrity, conformational changes, and molecular recognition. Here we report studies of site-and sequence-specific hydration dynamics, using 2-aminopurine (Ap) as the intrinsic fluorescence probe and with femtosecond resolution. The dodecamer d[CGCA(Ap)ATTTGCG]2 was investigated, and we also examined the effect of a specific minor groove-binding drug, pentamidine, on hydration dynamics. Two time scales were observed: Ϸ1 ps (bulklike) and 10 -12 ps (weakly bound type), consistent with layer hydration observed in proteins and DNA. However, for denatured DNA, the cosolvent condition of 40% formamide hydration is very different: it becomes that of bulk (in the presence of formamide). Well known electron transfer between Ap and nearby bases in stacked assemblies becomes inefficient in the single-stranded state. The rigidity of Ap in the single strands is significantly higher than that in bulk water and that attached to deoxyribose, suggesting a unique role for the dynamics of the phosphate-sugarbase in helix formation. The disparity in minor and major groove hydration is evident because of the site selection of Ap and in the time scale observed here (in the presence and absence of the drug), which is different by a factor of 2 from that observed in the minor groove-drug recognition.T he influence of hydration on the conformation and interactions of DNA has been the subject of many investigations using x-ray crystallography, NMR, molecular dynamics, and thermodynamic techniques (for recent reviews see refs. 1-4). The important role of water in the three-dimensional structures adopted by DNA is summarized in one of these reviews (1). At the interface between DNA and proteins (5, 6), water molecules in the first hydration shell of DNA ''mark'' the positions where protein residues hydrogen-bond to DNA. Even for specificity of cleavage in DNA, water activity affects site-specific recognition of DNA by EcoRI (6) as it does in protein function (7).In the minor groove of a B-form DNA duplex, hydration and its role in drug binding are striking. In a recent publication (8) from this laboratory, we described how the drug (Hoechst 33258) bound in the minor groove of DNA can be used to probe the dynamics of the water layer. For a DNA dodecamer duplex d(CGCAAATTTGCG) 2 , two well separated hydration times were found: 1.4 and 19 ps, compared with 0.2 and 1.2 ps for the same drug in bulk water. For comparison, we also studied genomic calf thymus DNA for which the hydration exhibits time scales similar to those of the dodecamer DNA.In this study, we use a DNA base analog, 2-aminopurine (Ap), in the same dodecamer B-DNA duplex d(CGCAApATTTGCG) 2 as an intrinsic fluorescence probe. Ap has been shown by NMR spectroscopy and thermodynamic measurements to form a stable base pair with thymine in a DNA oligomer, stabilized by two hydrogen bonds in a Watson-Crick geometry (9, 10) (see Fig. 1). Thus it is reasonable to consider that the modification by Ap does not alte...

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“…The time-resolution of the set-up was estimated to be about 500 fs from the fits according to the procedure described in ref. 23. To reconstruct the time-resolved spectra, we followed the procedure described in ref.…”

Section: Time-resolved Spectroscopymentioning

confidence: 99%

“…They were fitted to a sum of two exponential components convoluted with a Gaussian function describing the instrument response function. 23 The fitting parameters are provided in Table 1. Those previously Fig.…”

Section: Excited-state Decay Kineticsmentioning

confidence: 99%

Ultrafast light-induced response of photoactive yellow protein chromophore analogues

Espagne

Paik

Changenet‐Barret

et al. 2007

Photochem Photobiol Sci

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The fluorescence decays of several analogues of the photoactive yellow protein (PYP) chromophore in aqueous solution have been measured by femtosecond fluorescence up-conversion and the corresponding time-resolved fluorescence spectra have been reconstructed. The native chromophore of PYP is a thioester derivative of p-coumaric acid in its trans deprotonated form. Fluorescence kinetics are reported for a thioester phenyl analogue and for two analogues where the thioester group has been changed to amide and carboxylate groups. The kinetics are compared to those we previously reported for the analogues bearing ketone and ester groups. The fluorescence decays of the full series are found to lie in the 1-10 ps range depending on the electron-acceptor character of the substituent, in good agreement with the excited-state relaxation kinetics extracted from transient absorption measurements. Steady-state photolysis is also examined and found to depend strongly on the nature of the substituent. While it has been shown that the ultrafast light-induced response of the chromophore in PYP is controlled by the properties of the protein nanospace, the present results demonstrate that, in solution, the relaxation dynamics and pathway of the chromophore is controlled by its electron donor-acceptor structure: structures of stronger electron donor-acceptor character lead to faster decays and less photoisomerisation.

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Femtosecond Charge Transfer Dynamics of a Modified DNA Base: 2-Aminopurine in Complexes with Nucleotides

Cited by 109 publications

References 38 publications

The RNA–protein complex: Direct probing of the interfacial recognition dynamics and its correlation with biological functions

The RNA–protein complex: Direct probing of the interfacial recognition dynamics and its correlation with biological functions

Site- and sequence-selective ultrafast hydration of DNA

Ultrafast light-induced response of photoactive yellow protein chromophore analogues

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