Prediction of protein crystallization outcome using a hybrid method

The Thermofluor assay, also referred to as a thermal shift assay or differential scanning fluorescence (DSF), is a fast and simple method that is based upon high-throughput measurements of protein stability. The Thermofluor method can be performed on nearly all qPCR machines, meaning no special instrumentation is necessary, and can be used to validate the quality of protein preparations, screen for ligands or cofactors, and discover buffers and additives that maximize protein stability. This unit describes how to set up a Thermofluor method on several common models of qPCR instruments, how to prepare the samples for the assay, and how to run and analyze the resulting data. The unit also describes a 96-well screen to determine optimal buffer conditions for protein stability, which may assist in protein crystallization, and details the use of custom software, developed specifically for the analysis of data from Thermofluor screens.

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“…observ.). Related observations have been reported by others (Niesen et al, 2007;Mezzasalma et al, 2007;Zucker et al, 2010).…”

Section: Preparation Of a Thermofluor Assay For Buffer Optimizationsupporting

confidence: 88%

The Combined Use of the Thermofluor Assay and ThermoQ Analytical Software for the Determination of Protein Stability and Buffer Optimization as an Aid in Protein Crystallization

Phillips

Peña

2011

CP Molecular Biology

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“…This idea was validated experimentally and led to the crystallization of bacteriophage T4 lysozyme after creating by mutagenesis an artificial homodimer [225]. A recent attractive tool for crystallization prediction combines experimentally characterized physico-chemical features and sequence-derived data from target proteins [226]. Note that most criteria of crystallizability are correlated with the fact that the target should have an enhanced structural stability, as amply confirmed by many successful crystallization projects based on this idea (see below).…”

Section: Predicting Likelihood Of Crystallizationmentioning

confidence: 90%

A historical perspective on protein crystallization from 1840 to the present day

Giegé

2013

FEBS J

106

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Protein crystallization has been known since 1840 and can prove to be straightforward but, in most cases, it constitutes a real bottleneck. This stimulated the birth of the biocrystallogenesis field with both ‘practical’ and ‘basic’ science aims. In the early years of biochemistry, crystallization was a tool for the preparation of biological substances. Today, biocrystallogenesis aims to provide efficient methods for crystal fabrication and a means to optimize crystal quality for X‐ray crystallography. The historical development of crystallization methods for structural biology occurred first in conjunction with that of biochemical and genetic methods for macromolecule production, then with the development of structure determination methodologies and, recently, with routine access to synchrotron X‐ray sources. Previously, the identification of conditions that sustain crystal growth occurred mostly empirically but, in recent decades, this has moved progressively towards more rationality as a result of a deeper understanding of the physical chemistry of protein crystal growth and the use of idea‐driven screening and high‐throughput procedures. Protein and nucleic acid engineering procedures to facilitate crystallization, as well as crystallization methods in gelled‐media or by counter‐diffusion, represent recent important achievements, although the underlying concepts are old. The new nanotechnologies have brought a significant improvement in the practice of protein crystallization. Today, the increasing number of crystal structures deposited in the Protein Data Bank could mean that crystallization is no longer a bottleneck. This is not the case, however, because structural biology projects always become more challenging and thereby require adapted methods to enable the growth of the appropriate crystals, notably macromolecular assemblages.

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“…Combining experimental measurements and protein sequence information (e.g. [93] ) is an interesting approach. However experimental characterisation requires purified protein, therefore predictions would be focused on crystal growth and not available for decision-making during initial target selection.…”

Section: Discussionmentioning

confidence: 99%

Computational approaches to selecting and optimising targets for structural biology

Overton

Barton

2011

Methods

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Highlights► Identifies key considerations in target selection and optimisation. ► Approaches to assign useful protein features and structure/function relationships. ► Comparison of latest crystallisation propensity predictors on nonredundant data. ► Discusses single point of reference target selection/optimisation resources. ► Guidance on using the SSPF Target Optimisation Utility (TarO).

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Prediction of protein crystallization outcome using a hybrid method

Cited by 18 publications

References 37 publications

The Combined Use of the Thermofluor Assay and ThermoQ Analytical Software for the Determination of Protein Stability and Buffer Optimization as an Aid in Protein Crystallization

The Combined Use of the Thermofluor Assay and ThermoQ Analytical Software for the Determination of Protein Stability and Buffer Optimization as an Aid in Protein Crystallization

A historical perspective on protein crystallization from 1840 to the present day

Computational approaches to selecting and optimising targets for structural biology

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