Design, Synthesis, and Study of Fluorinated Proteins

Abstract

Highly fluorinated analogs of hydrophobic amino acids have proven to be generally effective in increasing the thermodynamic stability of proteins. These non-proteogenic amino acids can be incorporated into both α-helix and β-sheet structural motifs and generally enhance protein stability towards unfolding by heat and chemical denaturants, and retard their degradation by proteases. Recent detailed structural and thermodynamic studies have demonstrated that the increase in buried hydrophobic surface area that accompanies fluorination is primarily responsible for the stabilizing properties of fluorinated side chains. Fluorination appears to be a particularly useful strategy for increasing protein stability because fluorinated amino acids closely retain the shape of the side chain, and are thus minimally perturbing to protein structure and function. The first part of this chapter discusses some examples of highly fluorinated model proteins designed by our laboratory and protocols for their synthesis. In the second part, methods for determining their thermodynamic stability, along with conditions that have proven to be useful for crystallizing these proteins, are presented.

Location dependent coordination chemistry and MRI relaxivity, in de novo designed lanthanide coiled coils

“…Chemical unfolding data was recorded by monitoring the ellipticity at 222 nm of a 30 μM solution of peptide monomer in 5 mM HEPES buffer pH 7.0 in the absence and presence of 10 μM GdCl 3 , as a function of urea concentration (from 0 → 6.5 M). The chemical denaturation data was fit to a two-state, folded to three monomers, equilibrium model using global analysis nonlinear least squares fitting in MATLAB as outlined in the procedure by Buer et al 34 Thermal unfolding experiments were recorded using a Jasco Peltier temperature accessory, over the temperature range 20–90 °C, with a temperature gradient of 0.38 °C min –1 , whilst monitoring the signal at 222 nm.…”

Protein stability: a crystallographer's perspective

“…In addition to alkylation, other chemical modifications such as fluorination can be carried out. Fluorine is all but absent from biological systems, but stabilizes proteins as a result of the 'fluorous effect' (Buer & Marsh, 2014;Marsh, 2014). This effect results in an unusual propensity to undergo phase separation and causes an increase in the buried surface area in the hydrophobic core of fluorinated proteins.…”

Synthesis of fluorinated leucines, valines and alanines for use in protein NMR

“…2 Due to favourable fluorous and hydrophobic interactions, substitution of canonical amino acids (AAs) with fluorinated analogues often results in increased thermodynamic stability and secondary structure propensity. 2,3 In some cases judicious fluorination increases the resilience of peptides and proteins towards proteolysis. 4 These effects have been exploited to improve thermal and catalytic properties of enzymes and the bioavailability of peptide therapeutics.…”