Cryo Conditions - Optimisation

Optimal buffer conditions

26237227 Haselbach NatMeth 2015

• Among the strongest determinants to preserve protein quaternary structure is a stringent control of solute pH. The stability maxima of many purified macromolecular complexes represent a broad pH range often far from neutrality. This conclusion is derived from the widely accepted observation that successful crystallization can only occur, if the protein under study is present as a thermodynamically stable, monodisperse species in solution
• DLS: prevalent opinion in the current literature is that the method is unsuitable for multi-domain proteins and multi-subunit macromolecular machines, on the basis that each individual domain/subunit would unfold independently giving rise to polyphasic unfolding curves¹⁰, ^12, ^13, yielding only information about the stability of individual components only. Therefore, as a starting point we initially determined if single – apparent two-state - unfolding transitions could be obtained with multi-domain proteins and/or multi-subunit macromolecular complexes and, importantly, if this behaviour correlated with sample monodispersity.

Reducing PfSnf2L size

pfSnf2L tends to form multimers that disappear in size exclusion. we have to make it smaller, stable, more defined.

Use inhibitors to de-multimerize

I-G3 creates the smallest DLS signals

(S2872 (5uM), GW5074 / gw5074) has the strongest effect. (nanoDSF 20uM)

I-G3 does not fully inhibit remodeling

next steps

titrate I-G3 - DLS; EMSA with nucleosomes in the presence of I-G3