Time-resolved crystallographic studies of light-induced structural changes in the photosynthetic reaction center

Baxter, Richard H. G.; Ponomarenko, Nina; Šrajer, V.; Pahl, R.; Moffat, Keith; Norris, James R.

doi:10.1073/pnas.0306840101

Cited by 66 publications

(76 citation statements)

References 42 publications

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“…Another noteworthy study applied time-resolved Laue diffraction to search for light-induced structural changes in a photosynthetic reaction centre, which marked the first application of this method to a membrane protein complex. In that case, however, no significant lightinduced changes in electron density changes were reported (Baxter et al, 2004). Despite the impressive technical gains achieved over recent years, the method of time-resolved Laue diffraction has not grown as rapidly over the last two decades as other challenging fields of structural biology such as membrane protein crystallography (White, 2009).…”

Section: Time-resolved Laue Diffractionmentioning

confidence: 99%

Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches

et al. 2010

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Proteins undergo conformational changes during their biological function. As such, a high-resolution structure of a protein's resting conformation provides a starting point for elucidating its reaction mechanism, but provides no direct information concerning the protein's conformational dynamics. Several X-ray methods have been developed to elucidate those conformational changes that occur during a protein's reaction, including time-resolved Laue diffraction and intermediate trapping studies on three-dimensional protein crystals, and timeresolved wide-angle X-ray scattering and X-ray absorption studies on proteins in the solution phase. This review emphasizes the scope and limitations of these complementary experimental approaches when seeking to understand protein conformational dynamics. These methods are illustrated using a limited set of examples including myoglobin and haemoglobin in complex with carbon monoxide, the simple light-driven proton pump bacteriorhodopsin, and the superoxide scavenger superoxide reductase. In conclusion, likely future developments of these methods at synchrotron X-ray sources and the potential impact of emerging X-ray free-electron laser facilities are speculated upon.

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Section: Time-resolved Laue Diffractionmentioning

confidence: 99%

Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches

et al. 2010

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“…We conducted a hybrid screen of RC (22 mg/ml/0.08% LDAO/7% heptane-triol/4.5% triethylammonium phosphate solution in 20 mM Na 2 HPO 4 /NaH 2 PO 4 buffer, pH 6.0) using a total of 20 reagent plugs of 18 precipitants (SI Table 3). In addition to 16 precipitants from the custom-made kit, we used 2 precipitants, both in duplicate plugs: one was a positive control based on the original crystallization condition (39,40) [4M (NH 4 ) 2 SO 4 in 50 mM Na 2 HPO 4 /NaH 2 PO 4 buffer pH 6.0], and another was a modified crystallization condition at higher pH [3.6 M (NH 4 ) 2 SO 4 in 50 mM Tris, pH 7.8]. The duplicates were placed at random positions within the array of precipitants.…”

Section: Testing Compatibility Of the Hybrid Methods With Crystallizatmentioning

confidence: 99%

Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

Li¹,

Mustafi²,

Fu³

et al. 2006

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

226

259

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High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a ''hybrid'' droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as Ϸ140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in Ϸ10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 l of protein solution, Ϸ1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.droplets ͉ plugs ͉ protein structure ͉ high-throughput ͉ miniaturization T his work reports a ''hybrid'' microfluidic approach that uses nanoliter plugs to perform screening and optimization simultaneously in the same experiment. To validate this method using a challenging problem, we demonstrate its compatibility with crystallization of membrane proteins. Small-scale screening and optimization experiments are important for biological assays, chemical screening, and protein crystallization (1-3). Screening and optimization are usually carried out sequentially. In the case of protein crystallization, random sparse matrix screening initially identifies the precipitants that may lead to crystallization. Subsequent gradient optimization establishes concentrations of these precipitants that lead to diffractionquality crystals (4). Combining screening and optimization steps into a single hybrid experiment would eliminate the need to wait for the outcome of the initial screen before carrying out subsequent optimizations. Furthermore, a hybrid experiment would reduce the false negatives (5) associated with screens performed at a single concentration. The hybrid experiment could also be more conclusive, because a single batch of the s...

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“…the reorientation of the Ser223 residue in the vicinity of the Q B binding site [26,27]. The latter conformational changes may likely be possible only upon activation of molecular dynamics as a "lubricant" [10,13].…”

Section: Dynamics-function Correlationmentioning

confidence: 99%

The functional role of protein dynamics in photosynthetic reaction centers investigated by elastic and quasielastic neutron scattering

Pieper

2015

EPJ Web of Conferences

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Abstract. This short review summarizes our current knowledge about the functional relevance of protein dynamics in photosynthetic reaction centers. In the case of Photosystem II membrane fragments, elastic and quasielastic neutron scattering experiments reveal a dynamical transition at about 240 K corresponding to the activation of picosecond molecular motions. Likewise, a "freezing" of molecular dynamics is observed upon dehydration. Intriguingly, these effects correlate with the pronounced temperature-and hydration-dependence of specific electron transfer steps in Photosystem II indicating that molecular dynamics is an indispensable prerequisite for its function. Thus, electron transfer in Photosystem II appears to be a prototypical example for a dynamics-function correlation. Finally, the laser-neutron pump-probe technique is shown to permit in-situ monitoring of molecular dynamics in specific functional states of a protein in real time.

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Time-resolved crystallographic studies of light-induced structural changes in the photosynthetic reaction center

Cited by 66 publications

References 42 publications

Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches

Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches

Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

The functional role of protein dynamics in photosynthetic reaction centers investigated by elastic and quasielastic neutron scattering

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