Binding of calcium — and small molecules in general — often induce conformational changes in large molecules and complexes. The degree and type of change varies, but the resulting shift in specific affinities ultimately induces a physiological response. It is therefore important for our understanding of responses at the cellular level to define coupled changes at the molecular level.
Calumenin, a six-EF-hand calcium-binding protein localized in the endoplasmic reticulum, undergoes substantial calcium-induced rearrangement. We have demonstrated how calumenin changes from being unfolded in the absence of calcium to a compact trilobal fold in the presence of calcium (Mazzorana et al., PLoS One 11:e0151547, 2016).
Here, we describe protocols for the expression and purification of calumenin and calmodulin, another EF-hand protein modulated by calcium, along with protocols for biophysical techniques used to characterize calcium-induced changes to protein conformation. Analytical size-exclusion chromatography in the presence and absence of calcium provides an informed indication of any larger conformational movements. Circular dichroism spectroscopy reveals alterations to the secondary or tertiary structure, while small-angle X-ray scattering explores changes further providing low-resolution conformational details.
Surface plasmon resonance estimates binding kinetics and affinities completing the biophysical description of these events.
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The authors would like to thank Rohanah Hussain, Tamas Javorfi, Robert Rambo, and Gemma Harris for expert help and advice.
Du X, Li Y, Xia YL et al (2016) Insights into protein-ligand interactions: mechanisms, models, and methods. Int J Mol Sci 17:144CrossRefGoogle Scholar
Berridge MJ (1997) Elementary and global aspects of calcium signalling. J Physiol 499(Pt 2):291–306CrossRefGoogle Scholar
Mazzorana M, Hussain R, Sorensen T (2016) Ca-dependent folding of human calumenin. PLoS One 11:e0151547CrossRefGoogle Scholar
Sahin E, Roberts CJ (2012) Size-exclusion chromatography with multi-angle light scattering for elucidating protein aggregation mechanisms. Methods Mol Biol 899:403–423CrossRefGoogle Scholar
Scarlett G, Siligardi G, Kneale GG (2015) Circular dichroism for the analysis of protein-DNA interactions. Methods Mol Biol 1334:299–312CrossRefGoogle Scholar
Drescher DG, Selvakumar D, Drescher MJ (2018) Analysis of protein interactions by surface Plasmon resonance. Adv Protein Chem Struct Biol 110:1–30CrossRefGoogle Scholar
Rambo RP, Tainer JA (2010) Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small-angle X-ray scattering. Curr Opin Struct Biol 20:128–137CrossRefGoogle Scholar
Iida S, Potter JD (1986) Calcium binding to calmodulin. Cooperativity of the calcium-binding sites. J Biochem 99:1765–1772CrossRefGoogle Scholar
Zhang M, Abrams C, Wang L et al (2012) Structural basis for calmodulin as a dynamic calcium sensor. Structure 20:911–923CrossRefGoogle Scholar
Asiani KR, Williams H, Bird L et al (2016) SilE is an intrinsically disordered periplasmic “molecular sponge” involved in bacterial silver resistance. Mol Microbiol 101:731–742CrossRefGoogle Scholar
Wright PE, Dyson HJ (2015) Intrinsically disordered proteins in cellular signalling and regulation. Nat Rev Mol Cell Biol 16:18–29CrossRefGoogle Scholar
Ullah R, Shah MA, Tufail S et al (2016) Activity of the human rhinovirus 3C protease studied in various buffers, additives and detergents solutions for recombinant protein production. PLoS One 11:e0153436CrossRefGoogle Scholar
Mossessova E, Lima CD (2000) Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol Cell 5:865–876CrossRefGoogle Scholar
Berrow NS, Alderton D, Sainsbury S et al (2007) A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res 35:e45CrossRefGoogle Scholar
Berrow NS, Alderton D, Owens RJ (2009) The precise engineering of expression vectors using high-throughput In-Fusion PCR cloning. Methods Mol Biol 498:75–90CrossRefGoogle Scholar
Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234CrossRefGoogle Scholar
Javorfi T, Hussain R, Myatt D et al (2010) Measuring circular dichroism in a capillary cell using the b23 synchrotron radiation CD beamline at diamond light source. Chirality 22(Suppl 1):E149–E153CrossRefGoogle Scholar
Bers DM, Patton CW, Nuccitelli R (1994) A practical guide to the preparation of Ca2+ buffers. Methods Cell Biol 40:3–29CrossRefGoogle Scholar
Sreerama N, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287:252–260CrossRefGoogle Scholar
Rambo RP, Tainer JA (2013) Accurate assessment of mass, models and resolution by small-angle scattering. Nature 496:477–481CrossRefGoogle Scholar
Kursula P (2014) Crystallographic snapshots of initial steps in the collapse of the calmodulin central helix. Acta Crystallogr D Biol Crystallogr 70:24–30CrossRefGoogle Scholar