Sub-millisecond Chain Collapse of the Escherichia coli Globin ApoHmpH

Zhu, Li; Kurt, Neşe; Choi, Jennifer; Lapidus, Lisa J.; Cavagnero, Silvia

doi:10.1021/jp400174e

Cited by 9 publications

(10 citation statements)

References 61 publications

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“…Our results are also supported by a recent study on the folding of the apo-form of the heme-binding domain of flavohemoglobin (apoHmpH) from Escherichia coli , a protein with a globin fold structurally analogous to h-apoMb [ 68 ]. ApoHmpH, which has a single-Trp at position 120 in the H-helix, forms a kinetic intermediate during folding on the 10-μs time scale.…”

Section: Discussionsupporting

confidence: 83%

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

2015

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In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.

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Section: Discussionsupporting

confidence: 83%

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

2015

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“…In the T-mixer the mixing time was~8 ms and dilution of denaturant was 100Â (10,11,13). In the serpentine mixer the mixing time was 300 ms and the dilution of both protein and denaturant was 5Â (6,14).…”

Section: Microfluidic Mixer Fabricationmentioning

confidence: 99%

Complex Pathways in Folding of Protein G Explored by Simulation and Experiment

Lapidus

Acharya

Schwantes

et al. 2014

Biophysical Journal

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The B1 domain of protein G has been a classic model system of folding for decades, the subject of numerous experimental and computational studies. Most of the experimental work has focused on whether the protein folds via an intermediate, but the evidence is mostly limited to relatively slow kinetic observations with a few structural probes. In this work we observe folding on the submillisecond timescale with microfluidic mixers using a variety of probes including tryptophan fluorescence, circular dichroism, and photochemical oxidation. We find that each probe yields different kinetics and compare these observations with a Markov State Model constructed from large-scale molecular dynamics simulations and find a complex network of states that yield different kinetics for different observables. We conclude that there are many folding pathways before the final folding step and that these paths do not have large free energy barriers.

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“…Another common theme in early folding events has been the structural heterogeneity evident in early intermediates. Millisecond timescale studies of cytochrome c, CF‐based sub‐millisecond work on TIM barrels and globins suggest that a distribution of states possibly with marginal stability may be in dynamic equilibrium. Extended and compact states were observed for cytochrome c and a TIM barrel using distance distributions from FRET.…”

Section: Major Findingsmentioning

confidence: 99%

Advances in turbulent mixing techniques to study microsecond protein folding reactions

et al. 2013

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Recent experimental and computational advances in the protein folding arena have shown that the readout of the one-dimensional sequence information into three-dimensional structure begins within the first few microseconds of folding. The initiation of refolding reactions has been achieved by several means, including temperature jumps, flash photolysis, pressure jumps and rapid mixing methods. One of the most commonly used means of initiating refolding of chemically-denatured proteins is by turbulent flow mixing with refolding dilution buffer, where greater than 99% mixing efficiency has been achieved within 10’s of microseconds. Successful interfacing of turbulent flow mixers with complementary detection methods, including time-resolved Fluorescence Spectroscopy (trFL), Förster Resonance Energy Transfer (FRET), Circular Dichroism (CD), Small-Angle X-ray Scattering (SAXS), Hydrogen Exchange (HX) followed by Mass Spectrometry (MS) and Nuclear Magnetic Resonance Spectroscopy (NMR), Infrared Spectroscopy (IR), and Fourier Transform IR Spectroscopy (FTIR), has made this technique very attractive for monitoring various aspects of structure formation during folding. Although continuous-flow (CF) mixing devices interfaced with trFL detection have a dead time of only 30 µs, burst-phases have been detected in this time scale during folding of peptides and of large proteins (e.g., CheY and TIM barrels). Furthermore, a major limitation of CF mixing technique has been the requirement of large quantities of sample. In this brief communication, we will discuss the recent flurry of activity in micromachining and microfluidics, guided by computational simulations, that are likely to lead to dramatic improvements in time resolution and sample consumption for CF mixers over the next few years.

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Sub-millisecond Chain Collapse of the Escherichia coli Globin ApoHmpH

Cited by 9 publications

References 61 publications

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

Complex Pathways in Folding of Protein G Explored by Simulation and Experiment

Advances in turbulent mixing techniques to study microsecond protein folding reactions

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