Effects of energy transfer and rotational diffusion on the fluorescence polarization of macromolecules

Weber, Gregorio; Anderson, Sonia R.

doi:10.1021/bi00829a050

Cited by 44 publications

(20 citation statements)

References 7 publications

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“…The measurement of segmental flexibility has a long history (see Ref. 23); in particular, emission anisotropy has a long history of revealing local motions (24,25). In addition, timeresolved methods have the advantage of quantifying both the angular extent and the rate of these motions (24,26,27).…”

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confidence: 99%

Rapid Segmental and Subdomain Motions of DNA Polymerase β

Kim¹,

Beard²,

Harvey³

et al. 2003

Journal of Biological Chemistry

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DNA polymerase (pol) ␤ is a two-domain DNA repair enzyme that undergoes structural transitions upon binding substrates. Crystallographic structures indicate that these transitions include movement of the amino-terminal 8-kDa lyase domain relative to the 31-kDa polymerase domain. Additionally, a polymerase subdomain moves toward the nucleotide-binding pocket after nucleotide binding, resulting in critical contacts between ␣-helix N and the nascent base pair. Kinetic and structural characterization of pol ␤ has suggested that these conformational changes participate in stabilizing the ternary enzyme-substrate complex facilitating chemistry. To probe the microenvironment and dynamics of both the lyase domain and ␣-helix N in the polymerase domain, the single native tryptophan (Trp-325) of wild-type enzyme was replaced with alanine, and tryptophan was strategically substituted for residues in the lyase domain (F25W/W325A) or near the end of ␣-helix N (L287W/W325A). Influences of substrate on the fluorescence anisotropy decay of these single tryptophan forms of pol ␤ were determined. The results revealed that the segmental motion of ␣-helix N was rapid (ϳ1 ns) and far more rapid than the step that limits chemistry. Binding of Mg 2؉ and/or gapped DNA did not cause a noticeable change in the rotational correlation time or angular amplitude of tryptophan in ␣-helix N. More important, binding of a correct nucleotide significantly limited the angular range of the nanosecond motion within ␣-helix N. In contrast, the segmental motion of the 8-kDa domain was "frozen" upon DNA binding alone, and this restriction did not increase further upon nucleotide binding. The dynamics of ␣-helix N are discussed from the perspective of the "open" to "closed" conformational change of pol ␤ deduced from crystallography, and the results are more generally discussed in the context of reaction cycle-regulated flexibility for proteins acting as molecular motors. DNA polymerase (pol)1 ␤ is a 39-kDa enzyme composed of two distinct domains of 8-and 31-kDa connected by a proteasehypersensitive hinge region. The amino-terminal 8-kDa domain possesses the 5Ј-deoxyribose phosphate lyase activity required to process the 5Ј-terminus in a DNA gap during base excision repair. The polymerase active site is part of the carboxyl-terminal 31-kDa domain (for a review see Ref. 1). The domain and subdomain organization of pol ␤ is illustrated in Fig. 1, A and B. Although pol ␤ appears to have evolved separately from other families of polymerases of known structure (2), it shares many general structural and mechanistic features with other polymerases. The polymerase domain is composed of three functionally distinguishable subdomains. The polymerase catalytic subdomain coordinates two divalent metal cations that assist the nucleotidyl transferase reaction. Two additional subdomains that have primary roles in duplex DNA binding and nascent base pair (nucleoside 5Ј-triphosphate and templating nucleotide) binding border the catalytic subdomain. These subdomains will ...

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confidence: 99%

Rapid Segmental and Subdomain Motions of DNA Polymerase β

Kim¹,

Beard²,

Harvey³

et al. 2003

Journal of Biological Chemistry

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“…Even if sufficient information is not available to calculate exact distances, relative dimensions of molecular structures can often be deduced (1,2,11), and in principle FRET is applicable to very complex molecular structures. The method has been applied successfully to estimate intra(inter)molecular distances between donor and acceptor molecules in biological molecules other than nucleic acids (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26), to determine molecular distributions in condensed media (27)(28)(29)(30)(31), and to estimate distances and distributions in a large variety of other polymer problems (26,32).…”

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confidence: 99%

Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer.

Clegg

Murchie²,

Zechel

et al. 1993

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

274

260

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The efficiency of fluorescence resonance energy transfer (FRET) between fluorescein and rhodamine covalently attached to both 5' termini of a series of doublestranded DNA species (ranging from 8 to 20 bp) was measured. FRET efficiency varied with a dependence compatible with dye-to-dye distances (R) calculated on the basis of doublestranded B-DNA structure; the helical geometry of doublestranded DNA in solution is clearly evident. The experimental data were consistent with a 1/[1 + (RIRo)6] dependence of FRET efficiency characteristic for the Forster dipole-dipole mechanism. The thermal dissociation of the strands of the duplex DNA species can be followed by using FRET, and from these data we have been able to obtain enthalpies of duplex formation in good agreement with earlier measurements using alternative techniques. FRET measurements at very different salt concentrations can be accurately compared. We conclude that FRET is a reliable and valuable method for studying structure and conformational transitions in nucleic acids.Nucleic acids may adopt specific and sometimes complex folded structures that are critical for their biological function. Full determination of these structures requires the measurement of distances up to 80 A or more, but there are few techniques that allow such distances to be determined in solution. This is particularly important for nucleic acids in view of the extended helical structures involved, which can lead to underdetermination of structures by methods that can only yield short distances. Fluorescence resonance energy transfer (FRET) is sensitive to distances in the longer size range and has recently proved to be very useful in the study of nucleic acid structures, such as the solution structure of the four-way DNA junction (1, 2). FRET-derived distance information in DNA and RNA could be complementary to the shorter distances determined by NMR. However, the application of FRET to fluorescence probes covalently linked to nucleic acid structures has been relatively infrequent (1,(3)(4)(5)(6)(7)(8)(9)(10)(11).The rate of nonradiative energy transfer from an excited donor molecule (D) to a nearby acceptor molecule (A) depends in a characteristic manner on the distance between the two chromophores and their relative angular disposition (12)(13)(14). Depending on the D-A molecular pairs, the efficiency of transfer responds sensitively to relatively small changes of distances in the range of 10 to 80 A. Careful evaluations of FRET experiments can yield quantitative estimates of distances between labeled positions in macromolecules, or in molecular aggregates, provided that certain spectroscopic parameters are known. Even if sufficient information is not available to calculate exact distances, relative dimensions of molecular structures can often be deduced (1, 2, 11), and in principle FRET is applicable to very complex molecular structures. The method has been applied successfully to estimate intra(inter)molecular distances between donor and acceptor molecules in biological mo...

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“…Thus, the T2 samples were not homogeneous and contained both monomolecular hairpins and bimolecular complexes. The fraction of the bimolecular species estimated according to Weber et al (13) was found to be 0.6±0.1.…”

Section: Results and Disscussionmentioning

confidence: 84%

“…The rotational relaxation time of the molecule is proportional to its hydrodynamic volume and molecular weight (13).…”

Section: Results and Disscussionmentioning

confidence: 99%

Structural Polymorphism of Oligo(dC) with Mixed α,β-Anomeric Backbone

Timofeev

Borisova

Shchyolkina

2000

Journal of Biomolecular Structure and Dynamics

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Oligonucleotides with mixed alpha,beta-anomeric backbone have been proposed recently for the recognition of random DNA sequence via new triplex motif (Doronina and Behr, Chem. Soc. Reviews 26, 63-71 (1997)). In the present work we examined alpha- and beta- anomers of cytidine as possible candidates to recognize AT and TA base pairs of the double stranded DNA. The binding properties of beta-oligo(dC) were studied on a series of synthetic oligodeoxynucleotides by UV absorbtion spectroscopy, measurements of bound EtBr fluorescence polarization, circular dichroism (CD) and non-denaturing gel electrophoresis. The UV thermal denaturation, polarization studies and CD experiments with three stranded oligonucleotide 5'-((dCalpha) (dCbeta))5-L-(dAT)5-L-(dAT)5 (L = triethyleneglycol linker) and other oligonucleotide models showed that the formation of semiprotonated oligocytidilic complexes takes place at low temperatures and neutral pH, rather than folding of the clip into intramolecular triplex. The low-temperature transition was observed in denaturation profiles of any oligonucleotide containing beta- or mixed alpha,beta- cytidine stretches at the concentration of 1 microM. Self-association of alpha,beta-oligo(dC) was additionally confirmed by the appearance of two CD bands (at 290 and 265 nm) characteristic of CC+ base pairs. Despite the effective ability of alpha,beta-oligo(dC) to form self-associates, we succeeded in targeting 30-bp AT containing random DNA duplex by a 30-nt alpha,beta-oligocytidilate as evidenced by non-denaturing gel electrophoresis. A complete binding of the duplex was observed at a 5-fold excess of the third strand at 15 degrees C. Along with the formation of the three-stranded complex, self-association of mixed backbone oligo(dC) strands occurred.

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Effects of energy transfer and rotational diffusion on the fluorescence polarization of macromolecules

Cited by 44 publications

References 7 publications

Rapid Segmental and Subdomain Motions of DNA Polymerase β

Rapid Segmental and Subdomain Motions of DNA Polymerase β

Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer.

Structural Polymorphism of Oligo(dC) with Mixed α,β-Anomeric Backbone

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