How fluorescent labelling alters the solution behaviour of proteins.

Physical chemistry chemical physics : PCCP (2015-11-07)
M K Quinn, N Gnan, S James, A Ninarello, F Sciortino, E Zaccarelli, J J McManus
ABSTRACT

A complete understanding of the role of molecular anisotropy in directing the self assembly of colloids and proteins remains a challenge for soft matter science and biophysics. For proteins in particular, the complexity of the surface at a molecular level poses a challenge for any theoretical and numerical description. A soft matter approach, based on patchy models, has been useful in describing protein phase behaviour. In this work we examine how chemical modification of the protein surface, by addition of a fluorophore, affects the physical properties of protein solutions. By using a carefully controlled experimental protein model (human gamma-D crystallin) and numerical simulations, we demonstrate that protein solution behaviour defined by anisotropic surface effects can be captured by a custom patchy particle model. In particular, the chemical modification is found to be equivalent to the addition of a large hydrophobic surface patch with a large attractive potential energy well, which produces a significant increase in the temperature at which liquid-liquid phase separation occurs, even for very low fractions of fluorescently labelled proteins. These results are therefore directly relevant to all applications based on the use of fluorescent labelling by chemical modification, which have become increasingly important in the understanding of biological processes and biophysical interactions.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium hydroxide, anhydrous, free-flowing, Redi-Dri, reagent grade, ≥98%, pellets
Sigma-Aldrich
Sodium hydroxide, ultra dry, powder or crystals, 99.99% trace metals basis
Sigma-Aldrich
Sodium hydroxide, reagent grade, 97%, flakes
Sigma-Aldrich
N,N-Dimethylformamide, biotech. grade, ≥99.9%
Sigma-Aldrich
Magnesium sulfate heptahydrate, ACS reagent, ≥98%
Sigma-Aldrich
N,N-Dimethylformamide, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99.8% (GC)
Sigma-Aldrich
Hydrochloric acid, 37 wt. % in H2O, 99.999% trace metals basis
Sigma-Aldrich
Hydrogen chloride solution, 4.0 M in dioxane
Sigma-Aldrich
Hydrogen chloride solution, 2.0 M in diethyl ether
Sigma-Aldrich
Sodium hydroxide, beads, 20-40 mesh, reagent grade, 97%
Sigma-Aldrich
Sodium hydroxide solution, 5.0 M
Sigma-Aldrich
Magnesium sulfate heptahydrate, ReagentPlus®, ≥99.0%
Sigma-Aldrich
Sodium hydroxide, puriss. p.a., ACS reagent, K ≤0.02%, ≥98.0% (T), pellets
Sigma-Aldrich
Magnesium sulfate heptahydrate, 99.5-100.5% (calc. to the dried substance), meets analytical specification of Ph. Eur., BP,USP, FCC
Sigma-Aldrich
Sodium hydroxide, reagent grade, 97%, powder
Sigma-Aldrich
Magnesium sulfate heptahydrate, puriss. p.a., ACS reagent, ≥99.0% (KT)
Sigma-Aldrich
Sodium hydroxide, reagent grade, ≥98%, pellets (anhydrous)
Sigma-Aldrich
Hydrogen chloride solution, 1.0 M in acetic acid
Sigma-Aldrich
Hydrochloric acid, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., fuming, ≥37%, APHA: ≤10
Sigma-Aldrich
Sodium hydroxide, ACS reagent, ≥97.0%, pellets
Sigma-Aldrich
Hydrogen chloride solution, 1.0 M in diethyl ether
Sigma-Aldrich
Hydrochloric acid, ACS reagent, 37%
Sigma-Aldrich
Sodium hydroxide, pellets, semiconductor grade, 99.99% trace metals basis
Sigma-Aldrich
N,N-Dimethylformamide, ACS reagent, ≥99.8%
Sigma-Aldrich
Sodium hydroxide solution, 50% in H2O
Sigma-Aldrich
N,N-Dimethylformamide, ReagentPlus®, ≥99%
Sigma-Aldrich
Hydrochloric acid, ACS reagent, 37%
Sigma-Aldrich
Sodium hydroxide, puriss. p.a., ACS reagent, reag. Ph. Eur., K ≤0.02%, ≥98%, pellets
Sigma-Aldrich
Sodium hydroxide, BioXtra, ≥98% (acidimetric), pellets (anhydrous)
Sigma-Aldrich
Hydrochloric acid, meets analytical specification of Ph. Eur., BP, NF, fuming, 36.5-38%