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Methods in Molecular Biology

DOI: 10.1007/978-1-4939-2230-7_14 DOI Help
PMID: 25502204 PMID Help

Authors: Giuliano Siligardi (Diamond Light Source) , Rohanah Hussain (Diamond Light Source)
Co-authored by industrial partner: No

Type: Book Chapter

State: Published (Approved)
Published: February 2015
Diamond Proposal Number(s): 8714 , 8893

Open Access Open Access

Abstract: The production of diffraction quality protein crystals for X-ray crystallography has been greatly accelerated by the development of high-throughput protein (HTP) methods, which enable a large number of crystallization conditions to be rapidly investigated. Monitoring sample quality and the effect of crystallization buffers on protein behavior in solution should be considered as part of the crystallization experiment. Circular Dichroism (CD) spectroscopy is the ideal technique for these tasks as it can be operated in a high-throughput mode. Using CD to screen ligand binding interactions could show whether protein function/activity is retained, altered, or lost under different crystallization conditions. In this chapter, several methods for high-throughput CD (HTCD) applied to the preparation of proteins for crystallization will be presented. Quality control (QC) of protein batches in terms of conformational folding is often disregarded in protein production. Examples of batch-to-batch variation in the local tertiary structure of aromatic side chain residues revealed by CD will be discussed. In some of the examples, the fact that ligand binding properties were affected by changes in folding clearly shows that the characterization of folding of recombinant protein batches should not be ignored but be implemented as an important part of protein quality control.

Journal Keywords: Circular Dichroism (Cd); Synchrotron Radiation Circular Dichroism (SRCD); Protein Secondary Structure; Local Tertiary Structure; High-Throughput Cd (Htcd); SRCD Uv-Denaturation Assay

Subject Areas: Biology and Bio-materials, Technique Development, Medicine


Instruments: B23-Circular Dichroism