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

Graceffa, Rita; Nobrega, R. Paul; Barrea, Raúl A.; Kathuria, Sagar V.; Chakravarthy, Srinivas; Bilsel, Osman; Irving, Thomas C.

doi:10.1107/s0909049513021833

Cited by 65 publications

(43 citation statements)

References 32 publications

(44 reference statements)

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“…66 The experimental details of sub-millisecond kinetics measured using the micro-SAXS setup have been previously described. 64 The final protein concentration in both setups was 2 mg•mL −1 .…”

Section: Methodsmentioning

confidence: 99%

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

Kathuria

Kayatekin

Barrea

et al. 2014

Journal of Molecular Biology

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It is generally held that random coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully-folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59 to heme distance was probed by time-resolved Förster resonance energy transfer (trFRET) in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59-heme distance close to that of the GdnHCl denatured state is present after ~27 µs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET state (efficiency > 90%) show that the collapse is barrier-limited. Small-angle x-ray scattering measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native like conditions.

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Section: Methodsmentioning

confidence: 99%

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

Kathuria

Kayatekin

Barrea

et al. 2014

Journal of Molecular Biology

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“…SAXS data can provide information about the structure, shape and size of chromatin and its complexes with interacting proteins. A new development, time-resolved SAXS can potentially be utilized to monitor real-time conformational changes induced by chromatin binding proteins (Graceffa, Nobrega, Barrea, Kathuria, Chakravarthy, Bilsel, et al, 2013;Kathuria, Kayatekin, Barrea, Kondrashkina, Graceffa, Guo, et al, 2014). …”

Section: Quality Control Strategies For In Vitro Reconstituted Chrmentioning

confidence: 99%

In Vitro Chromatin Assembly

Muthurajan

Mattiroli

Bergeron

et al. 2016

Methods in Enzymology

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Chromatin accessibility is modulated by structural transitions that provide timely access to the genetic and epigenetic information during many essential nuclear processes. These transitions are orchestrated by regulatory proteins that coordinate intricate structural modifications and signalling pathways. In vitro reconstituted chromatin samples from defined components are instrumental in defining the mechanistic details of such processes. The bottleneck to appropriate in vitro analysis is the production of high quality, and quality-controlled, chromatin substrates. In this chapter we describe methods for in vitro chromatin reconstitution and quality control. We highlight the strengths and weaknesses of various approaches, and emphasize quality control steps that ensure reconstitution of a bona fide homogenous chromatin preparation. This is essential for optimal reproducibility and reliability of ensuing experiments using chromatin substrates.

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“…CF-SAXS measurements were made as previously described (29). The total flow rate was 20 mL·min −1 using a 1:10 dilution of the unfolded protein for a final protein concentration of 1.5 mg·mL −1 in 10 mM potassium phosphate, 8 M urea at pH 7.0 and refolding with 0 M urea buffer.…”

Section: Methodsmentioning

confidence: 99%

Modulation of frustration in folding by sequence permutation

Nobrega

Arora

Kathuria

et al. 2014

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

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Folding of globular proteins can be envisioned as the contraction of a random coil unfolded state toward the native state on an energy surface rough with local minima trapping frustrated species. These substructures impede productive folding and can serve as nucleation sites for aggregation reactions. However, little is known about the relationship between frustration and its underlying sequence determinants. Chemotaxis response regulator Y (CheY), a 129-amino acid bacterial protein, has been shown previously to populate an offpathway kinetic trap in the microsecond time range. The frustration has been ascribed to premature docking of the N-and C-terminal subdomains or, alternatively, to the formation of an unproductive local-in-sequence cluster of branched aliphatic side chains, isoleucine, leucine, and valine (ILV). The roles of the subdomains and ILV clusters in frustration were tested by altering the sequence connectivity using circular permutations. Surprisingly, the stability and buried surface area of the intermediate could be increased or decreased depending on the location of the termini. Comparison with the results of small-angle X-ray-scattering experiments and simulations points to the accelerated formation of a more compact, on-pathway species for the more stable intermediate. The effect of chain connectivity in modulating the structures and stabilities of the early kinetic traps in CheY is better understood in terms of the ILV cluster model. However, the subdomain model captures the requirement for an intact N-terminal domain to access the native conformation. Chain entropy and aliphatic-rich sequences play crucial roles in biasing the early events leading to frustration in the folding of CheY.CF-SAXS | Go models | CheY permutants | protein-folding intermediates H ighly denatured states of globular proteins resemble statistical random coils when examined with low-resolution techniques such as X-ray scattering (1) and hydrodynamic analyses (2). However, a higher-resolution view provided by experimental models (3-6) and simulations (7) shows that the conformational ensemble is biased toward low-contact-order (CO) structures, e.g., α-helices, β-turns, and β-hairpins, which form and melt in less than a few microseconds. During folding, these nascent structures presumably coalesce into higher-order assemblies of ever-increasing free energy until reaching the transition-state ensemble (TSE) that leads to the native conformation. From another perspective, this assembly process mediates a global collapse of the chain in an unfavorable solvent (8). Landscape theory (9) posits that, in the simplest scenario, native-like substructures appear and lead without pause to the TSE and the native conformation in an apparent two-state fashion. However, simulations have found that topological frustration, e.g., the premature formation of a substructure that impedes access to the productive TSE, can lead to the accumulation of intermediates (I) that must unfold to some extent to traverse the folding reaction coordinate...

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

Cited by 65 publications

References 32 publications

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

In Vitro Chromatin Assembly

Modulation of frustration in folding by sequence permutation

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