Dynamic Light Scattering in Protein Crystallization Droplets: Adaptations for Analysis and Optimization of Crystallization Processes

Dierks, Karsten; Meyer, Arne; Einspahr, Howard; Betzel, Christian

doi:10.1021/cg701067r

Cited by 40 publications

(42 citation statements)

References 18 publications

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“…Another tool for routine analysis, scoring and optimization of the crystallization processes is dynamic light scattering (DLS). It has been demonstrated that the rate of the particle size growth is a good predictor of the outcome for the crystallization experiment and allows to assess the probability of obtaining macromolecule crystals before their growth starts [234]. In the evaluation of the crystallization trials it is also possible to utilize the fluorescence of tryptophan residues under UV light.…”

Section: Real-time Monitoring Of Crystalsmentioning

confidence: 99%

An Overview of Biological Macromolecule Crystallization

Krauss

Merlino

Vergara

et al. 2013

IJMS

127

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The elucidation of the three dimensional structure of biological macromolecules has provided an important contribution to our current understanding of many basic mechanisms involved in life processes. This enormous impact largely results from the ability of X-ray crystallography to provide accurate structural details at atomic resolution that are a prerequisite for a deeper insight on the way in which bio-macromolecules interact with each other to build up supramolecular nano-machines capable of performing specialized biological functions. With the advent of high-energy synchrotron sources and the development of sophisticated software to solve X-ray and neutron crystal structures of large molecules, the crystallization step has become even more the bottleneck of a successful structure determination. This review introduces the general aspects of protein crystallization, summarizes conventional and innovative crystallization methods and focuses on the new strategies utilized to improve the success rate of experiments and increase crystal diffraction quality.

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Section: Real-time Monitoring Of Crystalsmentioning

confidence: 99%

An Overview of Biological Macromolecule Crystallization

Krauss

Merlino

Vergara

et al. 2013

IJMS

127

View full text Add to dashboard Cite

show abstract

“…The inset in d shows a selected magnification such as Ca 2+ and Mg 2+ at concentration of 10 mM, were determined by DLS. The DLS system (SPECTROLIGHT™ 600, Xtal Concepts, Hamburg, Germany) was also used to monitor self-assembly in a corresponding 3 mg mL −1 solution after addition of different bivalent cations in situ (Dierks et al 2008). All DLS measurements were carried out at room temperature and evaluated using the provided software.…”

Section: Methodsmentioning

confidence: 99%

Analysis of self-assembly of S-layer protein slp-B53 from Lysinibacillus sphaericus

Drobot

Oberthüer

et al. 2016

Eur Biophys J

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The formation of stable and functional surface layers (S-layers) via self-assembly of surface-layer proteins on the cell surface is a dynamic and complex process. S-layers facilitate a number of important biological functions, e.g., providing protection and mediating selective exchange of molecules and thereby functioning as molecular sieves. Furthermore, S-layers selectively bind several metal ions including uranium, palladium, gold, and europium, some of them with high affinity. Most current research on surface layers focuses on investigating crystalline arrays of protein subunits in Archaea and bacteria. In this work, several complementary analytical techniques and methods have been applied to examine structure-function relationships and dynamics for assembly of S-layer protein slp-B53 from Lysinibacillus sphaericus: (1) The secondary structure of the S-layer protein was analyzed by circular dichroism spectroscopy; (2) Small-angle X-ray scattering was applied to gain insights into the three-dimensional structure in solution; (3) The interaction with bivalent cations was followed by differential scanning calorimetry; (4) The dynamics and time-dependent assembly of S-layers were followed by applying dynamic light scattering; (5) The two-dimensional structure of the paracrystalline S-layer lattice was examined by atomic force microscopy. The data obtained provide essential structural insights into the mechanism of S-layer self-assembly, particularly with respect to binding of bivalent cations, i.e., Mg and Ca. Furthermore, the results obtained highlight potential applications of S-layers in the fields of micromaterials and nanobiotechnology by providing engineered or individual symmetric thin protein layers, e.g., for protective, antimicrobial, or otherwise functionalized surfaces.

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“…The reservoirs were filled with 35 µL protein buffer (20 mM MOPS, pH 8, 1% beta-OG) and the plate was sealed with SmartSeal (Greiner Bio-One GmbH, Germany) to avoid evaporation during the measurements. DLS measurements within these droplets were carried out at 20°C using a Spectro Light 500 instrument (XtalConcepts GmbH, Germany), a plate reader for UV/visible-imaging and in situ DLS [52]. Data were analyzed using Spectro (Nabitec GmbH, Germany).…”

Section: Methodsmentioning

confidence: 99%

Production, Purification and Characterization of Recombinant, Full-Length Human Claudin-1

et al. 2013

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The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.

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Dynamic Light Scattering in Protein Crystallization Droplets: Adaptations for Analysis and Optimization of Crystallization Processes

Cited by 40 publications

References 18 publications

An Overview of Biological Macromolecule Crystallization

An Overview of Biological Macromolecule Crystallization

Analysis of self-assembly of S-layer protein slp-B53 from Lysinibacillus sphaericus

Production, Purification and Characterization of Recombinant, Full-Length Human Claudin-1

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