An ignored variable: solution preparation temperature in protein crystallization

Chen, Ruiqing; Lu, Qi; Cheng, Qing-Di; Ao, Liang-Bo; Zhang, Chenyan; Hou, Hai; Liu, Yong-Ming; Li, Dawei; Yin, Da‐Chuan

doi:10.1038/srep07797

Cited by 18 publications

(9 citation statements)

References 27 publications

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“…It is also known that crystal packing interactions can impede ligand binding, 52 and that poor reproducibility of structures crystalized under the same conditions can occur. 53 However, to the best of our knowledge, there have been no reports of similar observations where ligand-free protein crystallized while in solution more than 99.9999% is ligand-bound. We attribute most of this finding to the catalytic effect of nucleation centers that change the crystallization kinetics.…”

Section: Resultsmentioning

confidence: 92%

“…The relationship between the structure observed in the crystal and the dominant structure in solution has been examined since the beginning of protein crystallography. It is also known that crystal packing interactions can impede ligand binding, and that poor reproducibility of structures crystallized under the same conditions can occur . However, to the best of our knowledge, there have been no reports of similar observations where ligand-free protein crystallized while in a solution that was more than 99.9999% ligand-bound.…”

Section: Resultsmentioning

confidence: 99%

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Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii

et al. 2016

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The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system in studies of the role of protein dynamics in catalysis, but so far these studies were limited by the availability of structural information. Second, CbFDH and its mutants are of use in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in its commercial development. Here, we report the crystallization and structural determination of both holo-CbFDH and apo-CbFDH. The free energy barrier for the catalyzed reaction is computed, and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: The apo-enzyme crystal was obtained under co-crystallization conditions with a saturating concentration of both the cofactor (NAD+) and inhibitor (azide), which has a nM dissociation constant. It was found that the fraction of the apo-enzyme present in the solution is less than 1.7x10−7 (i.e. the solution is 99.9999% holo-enzyme). This is an extreme case where the crystal structure represents an insignificant fraction of enzyme in solution, and a mechanism rationalizing this phenomenon is presented.

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii

et al. 2016

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“…In the initial screenings, the WT crystallized with crystallization success rates of 62% and 78% in the SER screening and in the ionic interactions screening, respectively. The screening preparation of the WT and the three mutants was conducted at a slightly lower ambient temperature in comparison to the initial screenings, which could have had a critical effect on the crystallization success rate . However, we were able to compare the crystallization characteristics of the mutants with those of the simultaneously crystallizing WT proteins since identical conditions prevailed.…”

Section: Resultsmentioning

confidence: 99%

Rational Crystal Contact Engineering of Lactobacillus brevis Alcohol Dehydrogenase To Promote Technical Protein Crystallization

Nowotny

Hermann

et al. 2019

Crystal Growth & Design

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Technical protein crystallization is an alternative to preparative chromatography for purification of proteins. However, only a few proteins are satisfactorily crystallizable for this technical purpose. In the present work, the crystallizability of Lactobacillus brevis alcohol dehydrogenase (LbADH) was significantly improved by rational engineering of its crystal contact patches. The concept was to exchange amino acids at the crystal contact patches with the objective of (i) surface entropy reduction (SER) and (ii) enhancement of ionic interactions. We present three newly designed, enzymatically active LbADH mutants with improved crystallizability: K32A (via SER) and Q126H and Q126K (both via enhancement of ionic interactions). The wild type crystallized with a low crystallization success rate in microbatch experiments. All mutants crystallized consistently with enhanced crystallization kinetics under identical conditions. Mutant K32A crystallized at reduced protein concentrations. Mutant Q126H crystallized at reduced concentrations of protein and the crystallization agent polyethylene glycol. Furthermore, the X-ray structure of mutant K32A reveals evidence of crystal contact enforcement which does explain enhanced crystallizability on the atomic level. The increased space–time yield of mutant K32A in stirred tank crystallizers demonstrates that rational crystal contact engineering is a powerful tool to promote technical protein crystallization.

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“…At present, we have not many investigations that deal with the role of temperature for protein crystallization [17][18][19][20][21], though we still find a few. Budayova-Spano et al, have developed new strategies and devices to grow big crystals for neutron diffraction [22], and for protein crystallization using buttons of dialysis [23,24].…”

Section: Introductionmentioning

confidence: 99%

Novel Devices for Transporting Protein Crystals to the Synchrotron Facilities and Thermal Protection of Protein Crystals

et al. 2018

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In this article, we use novel and non-conventional devices, based on polyolefins that help to increase the thermal protection of protein crystals in their crystallization conditions for crystallographic applications. The present contribution deals with the application of some ad hoc devices designed for transporting protein crystals to the synchrotron facilities. These new devices help transporting proteins without cryo-cooling them, therefore replacing the conventional dry Dewars. We crystallized four model proteins, using the classic sitting-drop vapor diffusion crystallization setups. The model proteins lysozyme, glucose isomerase, xylanase, and ferritin were used to obtain suitable crystals for high-resolution X-ray crystallographic research. Additionally, we evaluated the crystallization of apo-transferrin, which is involved in neurodegenerative diseases. As apo-transferrin is extremely sensitive to the changes in the crystallization temperature, we used it as a thermal sensor to prove the efficiency of these thermal protection devices when transporting proteins to the synchrotron facilities.

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An ignored variable: solution preparation temperature in protein crystallization

Cited by 18 publications

References 27 publications

Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii

Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii

Rational Crystal Contact Engineering of Lactobacillus brevis Alcohol Dehydrogenase To Promote Technical Protein Crystallization

Novel Devices for Transporting Protein Crystals to the Synchrotron Facilities and Thermal Protection of Protein Crystals

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