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A microfluidic approach for investigating the temperature dependence of biomolecular activity with single-molecule resolution.

ABSTRACT: We present a microfluidic approach for single-molecule studies of the temperature-dependent behavior of biomolecules, using a platform that combines microfluidic sample handling, on-chip temperature control, and total internal reflection fluorescence (TIRF) microscopy of surface-immobilized biomolecules. With efficient, rapid, and uniform heating by microheaters and in situ temperature measurements within a microfluidic flowcell by micro temperature sensors, closed-loop, accurate temperature control is achieved. To demonstrate its utility, the temperature-controlled microfluidic flowcell is coupled to a prism-based TIRF microscope and is used to investigate the temperature-dependence of ribosome and transfer RNA (tRNA) structural dynamics that are required for the rapid and precise translocation of tRNAs through the ribosome during protein synthesis. Our studies reveal that the previously characterized, thermally activated transitions between two global conformational states of the pre-translocation (PRE) ribosomal complex persist at physiological temperature. In addition, the temperature-dependence of the rates of transition between these two global conformational states of the PRE complex reveal well-defined, measurable, and disproportionate effects, providing a robust experimental framework for investigating the thermodynamic activation parameters that underlie transitions across these barriers.

SUBMITTER: Wang B

PROVIDER: S-EPMC3766768 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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A microfluidic approach for investigating the temperature dependence of biomolecular activity with single-molecule resolution.

Wang Bin B Ho Joseph J Fei Jingyi J Gonzalez Ruben L RL Lin Qiao Q

Lab on a chip 20101027 2

We present a microfluidic approach for single-molecule studies of the temperature-dependent behavior of biomolecules, using a platform that combines microfluidic sample handling, on-chip temperature control, and total internal reflection fluorescence (TIRF) microscopy of surface-immobilized biomolecules. With efficient, rapid, and uniform heating by microheaters and in situ temperature measurements within a microfluidic flowcell by micro temperature sensors, closed-loop, accurate temperature con ...[more]

PMID: 20981364

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Project description:The local Fourier-space relation between diffracted intensity I, diffraction wavevector q and dose D, , is key to probing and understanding radiation damage by X-rays and energetic particles in both diffraction and imaging experiments. The models used in protein crystallography for the last 50 years provide good fits to experimental I(q) versus nominal dose data, but have unclear physical significance. More recently, a fit to diffraction and imaging experiments suggested that the maximum tolerable dose varies as q -1 or linearly with resolution. Here, it is shown that crystallographic data have been strongly perturbed by the effects of spatially nonuniform crystal irradiation and diffraction during data collection. Reanalysis shows that these data are consistent with a purely exponential local dose dependence, = I 0(q)exp[-D/D e(q)], where D e(q) ? q ? with ? ? 1.7. A physics-based model for radiation damage, in which damage events occurring at random locations within a sample each cause energy deposition and blurring of the electron density within a small volume, predicts this exponential variation with dose for all q values and a decay exponent ? ? 2 in two and three dimensions, roughly consistent with both diffraction and imaging experiments over more than two orders of magnitude in resolution. The B-factor model used to account for radiation damage in crystallographic scaling programs is consistent with ? = 2, but may not accurately capture the dose dependencies of structure factors under typical nonuniform illumination conditions. The strong q dependence of radiation-induced diffraction decays implies that the previously proposed 20-30?MGy dose limit for protein crystallography should be replaced by a resolution-dependent dose limit that, for atomic resolution data sets, will be much smaller. The results suggest that the physics underlying basic experimental trends in radiation damage at T ? 100?K is straightforward and universal. Deviations of the local I(q, D) from strictly exponential behavior may provide mechanistic insights, especially into the radiation-damage processes responsible for the greatly increased radiation sensitivity observed at T ? 300?K.

Dataset Information

A microfluidic approach for investigating the temperature dependence of biomolecular activity with single-molecule resolution.

Publications

A microfluidic approach for investigating the temperature dependence of biomolecular activity with single-molecule resolution.

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