Unfolding kinetics of dimeric creatine kinase measured by stopped-flow small angle X-ray scattering

Zhu, Li; Qin, Zhan-Fen; Zhou, Jun‐Mei; Kihara, Hiroshi

doi:10.1016/j.biochi.2003.12.002

Cited by 6 publications

(3 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

Section: Introductionmentioning

confidence: 99%

“…SAXS is well suited to studying the folding-driven structural changes because it provides information about the global structure, even when the conformations are dynamic or irregular. 25 Time-resolved SAXS using stopped-flow mixers or continuous-flow microfluidic chambers have been used to study the kinetics of protein folding 26,27 and RNA folding 14,17,28 and virus assembly. 29 Taking advantage of a small volume mixer and fast X-ray detector, we show that the Azoarcus ribozyme exhibits multiple folding pathways with different kinetics and that partitioning between these pathways depends strongly on Mg 2+ concentration.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Multistage Collapse of a Bacterial Ribozyme Observed by Time-Resolved Small-Angle X-ray Scattering

et al. 2010

View full text Add to dashboard Cite

Ribozymes must fold into compact, native structures to function properly in the cell. The first step in forming the RNA tertiary structure is the neutralization of the phosphate charge by cations, followed by collapse of the unfolded molecules into more compact structures. The specificity of the collapse transition determines the structures of the folding intermediates and the folding time to the native state. However, the forces that enable specific collapse in RNA are not understood. Using time-resolved SAXS, we report that upon addition of 5 mM Mg 2+ to the Azoarcus group I ribozyme, up to 80% of chains form compact structures in less than 1 millisecond. In 1 mM Mg 2+ , the collapse transition produces extended structures that slowly approach the folded state, while ≥ 1.5 mM Mg 2+ leads to an ensemble of random coils that fold with multi-stage kinetics. Increased flexibility of molecules in the intermediate ensemble correlates with a Mg 2+ -dependent increase in the fast folding population and a previously unobserved crossover in the collapse kinetics. Partial denaturation of the unfolded RNA with urea also increases the fraction of chains following the fast-folding pathway. These results demonstrate that the preferred collapse mechanism depends on the extent of Mg 2+ -dependent charge neutralization, and that non-native interactions within the unfolded ensemble contribute to the heterogeneity of the ribozyme folding pathways at the very earliest stages of tertiary structure formation.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Multistage Collapse of a Bacterial Ribozyme Observed by Time-Resolved Small-Angle X-ray Scattering

et al. 2010

View full text Add to dashboard Cite

show abstract

“…Several detection methods such as spectroscopic measurements (1), NMR (2), circular dichroism (CD) (3 − 5), Raman scattering (6), microcalorimetry (7), size‐exclusion chromatography (8), and small‐angle X‐ray (9) and neutron (10, 11) scattering (SAXS and SANS) provide information on the conformational state of the biomolecules suspended in liquid media. Among the tools for probing protein conformational transitions or folding pathways, the spectroscopic measurements are perhaps the most popular.…”

Section: Introductionmentioning

confidence: 99%

Measurements of Label Free Protein Concentration and Conformational Changes Using a Microfluidic UV‐LED Method

Lee

Tripathi

2007

Biotechnology Progress

View full text Add to dashboard Cite

This paper presents a microchip-based system for measuring concentrations and dynamic conformational changes in proteins without any use of extrinsic fluorescent labeling. The microchannel flow of protein molecules was integrated with an ultraviolet light-emitting diode (UV-LED, lambda ex = 295 nm) and a photodetector (lambda em = 330 nm). The intrinsic fluorescence shift, arising from selectively exciting aromatic amino acid tryptophan (Trp), was monitored to quantify refolding pathways by dynamically varying the concentration of the chemical denaturant, urea. Short diffusion distances in the microchannel result in rapid equilibrium between protein and titrating solutions. Dilutions on the chip were tightly regulated using pressure controls, rather than syringe-based flow, as verified with extensive on-chip tracer dye controls. The concentrations of proteins were first measured using the UV-LED microfluidic platform, and the data showed detection limits down to 72, 128, and 250 nM for tryptophan, bovine serum albumin (BSA), and bovine carbonic anhydrase (BCA), respectively. To validate the protein assay method, folding transition experiments were performed using a well-characterized protein, BSA. The microchip protein refolding transitions using intrinsic fluorescence were well-correlated with conventional fluorometer experiments. The microfluidic platform facilitates refolding studies to identify rapidly the optimal folding strategy for a protein using small quantities of material. The technique offers a real alternative to bulky microfluidic systems consisting of large and expensive laser-based designs.

show abstract

Applications of Small Angle X-ray Scattering in Pharmaceutical Science

Boyd

Rades

2016

Advances in Delivery Science and Technology

View full text Add to dashboard Cite

Unfolding kinetics of dimeric creatine kinase measured by stopped-flow small angle X-ray scattering

Cited by 6 publications

References 16 publications

Multistage Collapse of a Bacterial Ribozyme Observed by Time-Resolved Small-Angle X-ray Scattering

Multistage Collapse of a Bacterial Ribozyme Observed by Time-Resolved Small-Angle X-ray Scattering

Measurements of Label Free Protein Concentration and Conformational Changes Using a Microfluidic UV‐LED Method

Applications of Small Angle X-ray Scattering in Pharmaceutical Science

Contact Info

Product

Resources

About