Cooperation between a salt bridge and the hydrophobic core triggers fold stabilization in a Trp-cage miniprotein.
Miniproteins are adequate models to study various protein-structure modifying effects such as temperature, pH, point mutation(s), H-bonds, salt bridges, molecular packing, etc. Tc5b, a 20-residue Trp-cage protein is one of the smallest of such models with a stable 3D fold (Neidigh J. W. et al. (2002) Nat. Struct. Biol. 9, 425-430). However, Tc5b exhibits considerable heat-sensitivity and is only stable at relatively low temperatures. Here we report a systematic investigation of structural factors influencing the stability of Tc5b by solving its solution structure in different environments, varying temperature, and pH. The key interactions identified are the hydrophobic stacking of the aromatic rings of Tyr3 and Trp6 and the salt bridge formed between Asp9 and Arg18. To verify the importance of these interactions, selected variants (mutated, glycosylated and truncated) of Tc5b were designed, prepared, and investigated by NMR. Indeed, elimination of either of the key interactions highly destabilizes the structure. These observations enabled us to design a new variant, Tc6b, differing only by a methylene group from Tc5b, in which both key interactions are optimized simultaneously. Tc6b exhibits enhanced heat stability and adopts a stable fold at physiological temperature.
Hudáky
Péter
P
Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös LorAnd University, PAzmAny Péter sétAny 1/A, H-1117 Budapest, Hungary.
Stráner
Pál
P
Farkas
Viktor
V
Váradi
Györgyi
G
Tóth
Gábor
G
Perczel
András
A
D000595:Amino Acid Sequence; D001216:Asparagine / Q000031:analogs & derivatives / Q000737:chemistry; D002942:Circular Dichroism; D005690:Galactose / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013696:Temperature
D010455:Peptides; C508725:Trp-cage peptide; D001216:Asparagine; D005690:Galactose