Electric-field-stimulated protein mechanics

Hekstra, Doeke R.; White, K. Ian; Socolich, Michael; Henning, Robert; Šrajer, V.; Ranganathan, Rama

doi:10.1038/nature20571

Cited by 188 publications

(235 citation statements)

References 54 publications

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“…Improving methods for reaction initiation by diffusion, discussed above, will open the door for time-resolved studies of a very large class of enzymatic reactions. In addition, the exciting new results point to another promising method of reaction initiation: electric field-stimulated crystallography (EF–X) (Hekstra et al ., 2016). …”

Section: Future Trends and Challengesmentioning

confidence: 99%

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Watching proteins function with time-resolved x-ray crystallography

Šrajer

Schmidt

2017

J. Phys. D: Appl. Phys.

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Macromolecular crystallography was immensely successful in the last two decades. To a large degree this success resulted from use of powerful third generation synchrotron X-ray sources. An expansive database of more than 100,000 protein structures, of which many were determined at resolution better than 2 Å, is available today. With this achievement, the spotlight in structural biology is shifting from determination of static structures to elucidating dynamic aspects of protein function. A powerful tool for addressing these aspects is time-resolved crystallography, where a genuine biological function is triggered in the crystal with a goal of capturing molecules in action and determining protein kinetics and structures of intermediates (Schmidt et al., 2005a; Schmidt 2008; Neutze and Moffat, 2012; Šrajer 2014). In this approach, short and intense X-ray pulses are used to probe intermediates in real time and at room temperature, in an ongoing reaction that is initiated synchronously and rapidly in the crystal. Time-resolved macromolecular crystallography with 100 ps time resolution at synchrotron X-ray sources is in its mature phase today, particularly for studies of reversible, light-initiated reactions. The advent of the new free electron lasers for hard X-rays (XFELs; 5–20 keV), which provide exceptionally intense, femtosecond X-ray pulses, marks a new frontier for time-resolved crystallography. The exploration of ultra-fast events becomes possible in high-resolution structural detail, on sub-picosecond time scales (Tenboer et al., 2014; Barends et al., 2015; Pande et al., 2016). We review here state-of-the-art time-resolved crystallographic experiments both at synchrotrons and XFELs. We also outline challenges and further developments necessary to broaden the application of these methods to many important proteins and enzymes of biomedical relevance.

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Section: Future Trends and Challengesmentioning

confidence: 99%

“…In the first EF–X time-resolved experiments conducted at BioCARS (Hekstra et al ., 2016), this new method for studying biomolecular machines was applied to the PDZ domain of the human E3 ubiquitin ligase LNX2. The protein was selected because it does not have known functional voltage dependence.…”

Section: Future Trends and Challengesmentioning

confidence: 99%

Watching proteins function with time-resolved x-ray crystallography

Šrajer

Schmidt

2017

J. Phys. D: Appl. Phys.

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“…In 2011, temperature jumps between 100 and 180 K were used to probe reaction intermediates in the photo-receptor protein, bacteriophytochrome, 61 and in 2016, electric field pulses were used to drive concerted motions within a crystal on the sub-μs timescale. 62 …”

Section: An Intertwined Historymentioning

confidence: 99%

X-rays in the Cryo-Electron Microscopy Era: Structural Biology’s Dynamic Future

Shoemaker

Ando

2018

Biochemistry

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Over the past several years, single-particle cryo-electron microscopy (cryo-EM) has emerged as a leading method for elucidating macromolecular structures at near-atomic resolution, rivaling even the established technique of X-ray crystallography. Cryo-EM is now able to probe proteins as small as hemoglobin (64 kDa), while avoiding the crystallization bottleneck entirely. The remarkable success of cryo-EM has called into question the continuing relevance of X-ray methods, particularly crystallography. To say that the future of structural biology is either cryo-EM or crystallography, however, would be misguided. Crystallography remains better suited to yield precise atomic coordinates of macromolecules under a few hundred kDa in size, while the ability to probe larger, potentially more disordered assemblies is a distinct advantage of cryo-EM. Likewise, crystallography is better equipped to provide high-resolution dynamic information as a function of time, temperature, pressure, and other perturbations, whereas cryo-EM offers increasing insight into conformational and energy landscapes, particularly as algorithms to deconvolute conformational heterogeneity become more advanced. Ultimately, the future of both techniques depends on how their individual strengths are utilized to tackle questions on the frontiers of structural biology. Structure determination is just one piece of a much larger puzzle: a central challenge of modern structural biology is to relate structural information to biological function. In this perspective, we share insight from several leaders in the field and examine the unique and complementary ways in which X-ray methods and cryo-EM can shape the future of structural biology.

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“…(Barends et al, 2015;Coquelle et al, 2018;Kern et al, 2018;Malmerberg et al, 2011;Nogly et al, 2018;Pande et al, 2016)). Unfortunately, the number of proteins that undergo specific photochemistry as part of their functional cycle is small, and there is a fundamental need to develop generalized methods that can be used to synchronously excite conformational transitions in any protein molecule and expand the utility of time-resolved structural experiments (Hekstra et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Temperature-Jump Solution X-ray Scattering Reveals Distinct Motions in a Dynamic Enzyme

Thompson

Barad

Wolff

et al. 2018

Preprint

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Correlated motions of proteins and their bound solvent molecules are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved methods hold promise for addressing this challenge but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature-jumps (T-jumps), through thermal excitation of the solvent, have been implemented to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform T-jump small-and wide-angle X-ray scattering (SAXS/WAXS) measurements on a dynamic enzyme, cyclophilin A (CypA), demonstrating that these experiments are able to capture functional intramolecular protein dynamics. We show that CypA displays rich dynamics following a T-jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes in the enzyme. Two relaxation processes are resolved, which can be characterized by Arrhenius behavior. We also used mutations that have distinct functional effects to disentangle the relationship of the observed relaxation processes. A fast process is related to surface loop motions important for substrate specificity, whereas a slower process is related to motions in the core of the protein that are critical for catalytic turnover. These results demonstrate the power of time-resolved X-ray scattering experiments for characterizing protein and solvent dynamics on the µs-ms timescale. We expect the T-jump methodology presented here will be useful for understanding kinetic correlations between local conformational changes of proteins and their bound solvent molecules, which are poorly explained by the results of traditional, static measurements of molecular structure.

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Electric-field-stimulated protein mechanics

Cited by 188 publications

References 54 publications

Watching proteins function with time-resolved x-ray crystallography

Watching proteins function with time-resolved x-ray crystallography

X-rays in the Cryo-Electron Microscopy Era: Structural Biology’s Dynamic Future

Temperature-Jump Solution X-ray Scattering Reveals Distinct Motions in a Dynamic Enzyme

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