Rescue of Misfolded Proteins and Stabilization by Small Molecules - Protein Misfolding and Cellular Stress in Disease and Aging - page 298

Chapter 22

Rescue of Misfolded Proteins and Stabilization by Small
Molecules

Raymond C. Stevens, Javier Sancho, and Aurora Martinez

Abstract

Increasing stability of functional proteins by binding small compounds and ions has long been used to
extend shelf-life of protein formulations in the pharmacological and biotechnological industry. Likewise,
the therapeutic application of small molecules for in vivo recovery and maintenance of structure and
function of proteins is steadily increasing. Compounds that can rescue misfolded proteins by stimulating
their correct folding and/or the stabilization of native-like conformations in vivo are referred to as phar-
macological chaperones. Here we present thermal-shift and isothermal methods for the high-throughput
screening of stabilizing pharmacological chaperones for soluble and membrane proteins. The effect of
selected hit compounds on the kinetics of protein synthesis is further evaluated by an in vitro transcrip-
tion-translation rapid translation system. These procedures can be integrated in an interdisciplinary and
translational approach for the search of personalized pharmacological chaperones in genetic misfolding
diseases.

Key words: Cytoplasmatic and membrane proteins, High-throughput, Experimental screening,
Protein stability, Misfolding diseases, Denaturation, Microscale fluorescence, Thermal-shift, Melting
temperature, Compound libraries

Abbreviations

ANS 8-Anilino 1-naphthalene sulfonic acid

C 0.5 Concentration for half-maximal binding

CPM N -[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide

DDM n -Dodecyl-b-D-maltoside

DMSO Dimethyl sulfoxide

GPCR G protein-coupled receptor

GnHCl Guanidinium hydrochloride

HTP High-throughput

λ em Emission wavelength

λ exc Excitation wavelength

PAH Phenylalanine hydroxylase

PKU Phenylketonuria

RTS Rapid translation system