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Studies on the structure and dynamics of the human telomeric G quadruplex by single-molecule fluorescence resonance energy transfer.

ABSTRACT: We have investigated the structure and unfolding kinetics of the human telomeric intramolecular G quadruplex by using single-molecule fluorescence resonance energy transfer. An exploration of conformational heterogeneity revealed two stable folded conformations, in both sodium- and potassium-containing buffers, with small differences between their enthalpies and entropies. Both folded conformations can be opened by the addition of a 21-base complementary DNA oligonucleotide. The unfolding of both substates occurs at the same rate, which showed dependence on the monovalent metal cation present. Temperature-dependence studies in 100 mM KCl gave an apparent activation enthalpy and entropy of 6.4 +/- 0.4 kcal.mol-1 and -52.3 +/- 1.4 cal.mol-1.K-1, respectively, indicating that the unfolding is entropically driven and can occur easily. In contrast, in 100 mM NaCl the respective values are 14.9 +/- 0.2 kcal.mol-1 and -23.0 +/- 0.8 cal.mol-1.K-1, suggesting a more significant enthalpic barrier. Molecular modeling suggests that the two species are likely to be the parallel and antiparallel quadruplex structures. The unfolding free energy barrier is estimated to be between 3 and 15 kBT based on Kramers' theory. We conclude that under near-physiological conditions these structures coexist and can interconvert on a minute time scale.

SUBMITTER: Ying L

PROVIDER: S-EPMC299749 | biostudies-literature | 2003 Dec

REPOSITORIES: biostudies-literature

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Studies on the structure and dynamics of the human telomeric G quadruplex by single-molecule fluorescence resonance energy transfer.

Ying Liming L Green Jeremy J JJ Li Haitao H Klenerman David D Balasubramanian Shankar S

Proceedings of the National Academy of Sciences of the United States of America 20031125 25

We have investigated the structure and unfolding kinetics of the human telomeric intramolecular G quadruplex by using single-molecule fluorescence resonance energy transfer. An exploration of conformational heterogeneity revealed two stable folded conformations, in both sodium- and potassium-containing buffers, with small differences between their enthalpies and entropies. Both folded conformations can be opened by the addition of a 21-base complementary DNA oligonucleotide. The unfolding of bot ...[more]

PMID: 14645716

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Project description:Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational phenotypes of interest.

Dataset Information

Studies on the structure and dynamics of the human telomeric G quadruplex by single-molecule fluorescence resonance energy transfer.

Publications

Studies on the structure and dynamics of the human telomeric G quadruplex by single-molecule fluorescence resonance energy transfer.

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