Monomeric green fluorescent protein as a protein standard for small angle scattering

Abstract

Abstract. Protein small angle scattering (SAS) has become increasing important in structural biochemistry, due to the increased performance and specification of new instruments and advances in the software and hardware used to analyse the data. Whilst all of this is encouraging, there is a lack of standardised experimental methodology within the community. Although a number of protein standards are currently used in SAS experiments to allow accurate molecular weight determination, each has specific advantages and disadvantages. We therefore propose the use of a mutated monomeric enhanced green fluorescent protein, as a protein standard, abbreviated to m-eGFP. It has a number of advantages over the currently used protein standards, for example it is cheap and easy to produce. It can be expressed in large amounts (>40 mg/L) in both hydrogenated and deuterated form. The mutation means it is highly monodisperse and GFP being a beta-barrel structure is thermodynamically stable over a number of days, giving highly reproducible results. We therefore believe m-eGFP is a good protein standard for small angle scattering (SAS).

Decorated networks of native proteins: nanomaterials with tunable mesoscopic domain size

“…Previous work on eGFP revealed that the protein exists in dimers. 46 When no YCl 3 is present, SAXS data indicates the presence of cylinders with a diameter of 30 Å and lengths of ∼80 Å, consistent with dimers as illustrated in the inset in Fig. 2a (Table SI, ESI † and Fig.…”

“…In order to determine these interactions we first fitted our SAXS data with a cylindrical form factor 46 to obtain information on the dimerisation and aggregation of small numbers of proteins. The results obtained are shown in Fig.…”

“…When the salt concentration is increased, we find a reduction of the strength of the repulsive interactions due to attractions between the proteins by either forming bridges (YCl 3 ) [30][31][32]43 or by decreasing their solubility through non-specific interactions ((NH 4 ) 2 SO 4 ). 29,45 In order to determine these interactions we first fitted our SAXS data with a cylindrical form factor 46 to obtain information on the dimerisation and aggregation of small numbers of proteins. The results obtained are shown in Fig.…”

Protein–polymer mixtures in the colloid limit: Aggregation, sedimentation, and crystallization

“…These results are in agreement with previous work on eGFP, where it was found that the protein exists in dimers. 103…”

Protein-Polymer Mixtures in the Colloid Limit: Aggregation, Sedimentation and Crystallization

“…These results are in agreement with previous work on eGFP, where it was found that the protein exists in dimers. 100 Electrophoretic Mobility Measurement. We performed electrophoretic mobility µ e measurements on 1 mL protein solutions of 2 mg/mL at 20 • C in a NaCl 10 mM solution using a Zetasizer Nano ZS (Malvern, UK) at a detector angle of 13 • and a 4 mW 633 nm laser beam to determine the charge of eGFP following Roosen-Runge, et al 91 Care was taken in order to have the same pH with the buffer used in phase diagram determination.…”