Microsecond Hydrophobic Collapse in the Folding of Escherichia coli Dihydrofolate Reductase, an α/β-Type Protein

Arai, Munehito; Kondrashkina, Elena; Kayatekin, Can; Matthews, C. Robert; Iwakura, Masahiro; Bilsel, Osman

doi:10.1016/j.jmb.2007.01.085

Cited by 79 publications

(96 citation statements)

References 63 publications

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“…The evolution of conformational changes of the products after mixing is probed by translating the microchannel horizontally (along the flow direction) across the X-ray beam and collecting the SAXS patterns. This data acquisition strategy is in contrast to earlier experiments where protein and blank scattering curves were acquired at one position along the channel prior to acquiring another time point (Arai et al, 2007;Wu et al, 2008). This process is very inefficient: the sample is continuously flowing to keep the steady-state flow condition (pressure variation of AE 1 p.s.i.…”

Section: Continuous Scanning Data Acquisitionmentioning

confidence: 99%

“…Small-solvent molecules and additives, such as chemical denaturants and metal ions, diffuse over this length scale within 10 ms. The plug flow in turbulent mixing gives rise to a relatively uniform reaction time in the channel orthogonal to the flow direction, making interfacing to SAXS relatively straightforward (Akiyama et al, 2002;Arai et al, 2007). Another advantage of turbulent mixers is that the linear flow velocity is typically $ 4 m s À1 (or $ 250 ms mm À1 ), resulting in a small dead-time ($ 150-200 ms).…”

Section: Introductionmentioning

confidence: 99%

“…Because of the high flow rates, sample can tolerate synchrotron X-ray flux without radiation damage and heating effects, delivering S/N comparable with those from static equilibrium experiments. In previously reported experiments, typical beam dimensions (35-50 mm FWMH in the vertical) limited time resolution by requiring relatively wide channels, $ 300 mm to avoid scattering from the tails of the beam hitting the edges of the channel (Arai et al, 2007;Wu et al, 2008). Better time resolution can be achieved using a smaller X-ray beam focal spot that would permit using narrower mixer channels.…”

Section: Introductionmentioning

confidence: 99%

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Sub-millisecond time-resolved SAXS using a continuous-flow mixer and X-ray microbeam

et al. 2013

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Small-angle X-ray scattering (SAXS) is a well established technique to probe the nanoscale structure and interactions in soft matter. It allows one to study the structure of native particles in near physiological environments and to analyze structural changes in response to variations in external conditions. The combination of microfluidics and SAXS provides a powerful tool to investigate dynamic processes on a molecular level with sub-millisecond time resolution. Reaction kinetics in the sub-millisecond time range has been achieved using continuous-flow mixers manufactured using micromachining techniques. The time resolution of these devices has previously been limited, in part, by the X-ray beam sizes delivered by typical SAXS beamlines. These limitations can be overcome using optics to focus X-rays to the micrometer size range providing that beam divergence and photon flux suitable for performing SAXS experiments can be maintained. Such micro-SAXS in combination with microfluidic devices would be an attractive probe for time-resolved studies. Here, the development of a high-duty-cycle scanning microsecond-timeresolution SAXS capability, built around the Kirkpatrick-Baez mirror-based microbeam system at the Biophysics Collaborative Access Team (BioCAT) beamline 18ID at the Advanced Photon Source, Argonne National Laboratory, is reported. A detailed description of the microbeam small-angle-scattering instrument, the turbulent flow mixer, as well as the data acquisition and control and analysis software is provided. Results are presented where this apparatus was used to study the folding of cytochrome c. Future prospects for this technique are discussed.

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Section: Continuous Scanning Data Acquisitionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Sub-millisecond time-resolved SAXS using a continuous-flow mixer and X-ray microbeam

et al. 2013

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“…Thus, time-resolved changes in the solution scattering pattern should provide a wealth of relevant information on protein structure and dynamics. The time resolution attainable in SAXS/WAXS studies had been limited to the millisecond timescale by the dead time for stopped-flow mixers (15), but with the advent of micromachined continuous-flow rapid mixers, it has been improved to ∼0.3 ms (16,17). Much shorter timescales can be accessed when the structural change is triggered by a laser pulse.…”

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confidence: 99%

Protein structural dynamics in solution unveiled via 100-ps time-resolved x-ray scattering

Cho

Dashdorj

Schotte

et al. 2010

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

142

156

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We have developed a time-resolved x-ray scattering diffractometer capable of probing structural dynamics of proteins in solution with 100-ps time resolution. This diffractometer, developed on the ID14B BioCARS (Consortium for Advanced Radiation Sources) beamline at the Advanced Photon Source, records x-ray scattering snapshots over a broad range of q spanning 0.02-2.5 Å −1 , thereby providing simultaneous coverage of the small-angle x-ray scattering (SAXS) and wide-angle x-ray scattering (WAXS) regions. To demonstrate its capabilities, we have tracked structural changes in myoglobin as it undergoes a photolysis-induced transition from its carbon monoxy form (MbCO) to its deoxy form (Mb). Though the differences between the MbCO and Mb crystal structures are small (rmsd <0.2 Å), time-resolved x-ray scattering differences recorded over 8 decades of time from 100 ps to 10 ms are rich in structure, illustrating the sensitivity of this technique. A strong, negative-going feature in the SAXS region appears promptly and corresponds to a sudden >22 Å 3 volume expansion of the protein. The ensuing conformational relaxation causes the protein to contract to a volume ∼2 Å 3 larger than MbCO within ∼10 ns. On the timescale for CO escape from the primary docking site, another change in the SAXS/WAXS fingerprint appears, demonstrating sensitivity to the location of the dissociated CO. Global analysis of the SAXS/WAXS patterns recovered time-independent scattering fingerprints for four intermediate states of Mb. These SAXS/WAXS fingerprints provide stringent constraints for putative models of conformational states and structural transitions between them.T o understand how a protein functions, it is crucial to know not only its high-resolution structure, but also how that structure evolves as it executes its designed function. To that end, we have developed time-resolved Laue methods capable of tracking structure changes in proteins with time resolution as short as 150 ps and spatial resolution better than 2 Å (1, 2). Like static structures, time-resolved structures are subject to crystal packing forces, which limit the range of conformational motion accessible to the protein. Indeed, the allosteric structure transition of human hemoglobin cannot be accommodated by the crystal; when individual molecules make that transition, macroscopic forces build up and crack the crystal (3). Clearly, techniques capable of probing protein conformational changes in solution are needed. Time-resolved spectroscopic techniques have long been used to probe dynamics of proteins in solution (4-9), but these measurements are sensitive primarily to the chromophore and its surrounding environment, and provide only indirect information regarding global structure changes. In contrast, x-ray scattering of proteins in solution produces 1D patterns that are sensitive to protein structure, with the so-called small-angle x-ray scattering (SAXS) region being sensitive to the size and shape of the protein (10-12), and the so-called wide-angle x-ray scattering (...

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“…For proteins with Ͼ100 residues, collapsed, partially folded intermediates are formed within hundreds of microseconds after the initiation of folding (1)(2)(3)(4). Quench-flow pulse-labeling experiments have yielded considerable information about the development of secondary structure in such intermediates, on a time scale of milliseconds (5,6).…”

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confidence: 99%

Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR

Uzawa

Nishimura

Akiyama

et al. 2008

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

172

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The earliest steps in the folding of proteins are complete on an extremely rapid time scale that is difficult to access experimentally. We have used rapid-mixing quench-flow methods to extend the time resolution of folding studies on apomyoglobin and elucidate the structural and dynamic features of members of the ensemble of intermediate states that are populated on a submillisecond time scale during this process. The picture that emerges is of a continuum of rapidly interconverting states. Even after only 0.4 ms of refolding time a compact state is formed that contains major parts of the A, G, and H helices, which are sufficiently well folded to protect amides from exchange. The B, C, and E helix regions fold more slowly and fluctuate rapidly between open and closed states as they search docking sites on this core; the secondary structure in these regions becomes stabilized as the refolding time is increased from 0.4 to 6 ms. No further stabilization occurs in the A, G, H core at 6 ms of folding time. These studies begin to timeresolve a progression of compact states between the fully unfolded and native folded states and confirm the presence an ensemble of intermediates that interconvert in a hierarchical sequence as the protein searches conformational space on its folding trajectory.protein folding ͉ pulse labeling ͉ rapid mixing M ost proteins fold rapidly from the highly heterogeneous conformational ensemble of the unfolded state into their well defined native conformations. For proteins with Ͼ100 residues, collapsed, partially folded intermediates are formed within hundreds of microseconds after the initiation of folding (1-4). Quench-flow pulse-labeling experiments have yielded considerable information about the development of secondary structure in such intermediates, on a time scale of milliseconds (5, 6). However, little is known about the processes that occur within the dead time (Ϸ6 ms) of the conventional quench flow apparatus, nor about the dynamic behavior of the kinetic folding intermediates. To gain insights into the early folding processes for sperm whale apomyoglobin, we have performed pulsed hydrogen-deuterium (H/D) exchange experiments with submillisecond time resolution.Apomyoglobin has been studied extensively by kinetic and equilibrium methods as a paradigm for understanding protein folding pathways and the structure of folding intermediates (7). The structure of apomyoglobin is similar to that of the holoprotein except that residues in the F helix and the C terminus of the H helix are disordered (8-10). During refolding, apomyoglobin forms an on pathway kinetic intermediate, in which major portions of the A, G, and H helices and part of the B helix are folded, within the 6-ms burst phase of conventional quench-flow H/D exchange experiments (11)(12)(13)(14). These same regions adopt stable secondary structure in the equilibrium molten globule intermediate formed at pH 4.2 (10,(15)(16)(17). Recent H/D exchange experiments under a variety of conditions detected heterogeneity in the apomyogl...

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Microsecond Hydrophobic Collapse in the Folding of Escherichia coli Dihydrofolate Reductase, an α/β-Type Protein

Cited by 79 publications

References 63 publications

Sub-millisecond time-resolved SAXS using a continuous-flow mixer and X-ray microbeam

Sub-millisecond time-resolved SAXS using a continuous-flow mixer and X-ray microbeam

Protein structural dynamics in solution unveiled via 100-ps time-resolved x-ray scattering

Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR

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