The denatured state of Engrailed Homeodomain under denaturing and native conditions.
Protein folding starts from the elusive form of the denatured state that is present under conditions that favour the native state. We have studied the denatured state of Engrailed Homeodomain (En-HD) under mildly and strongly denaturing conditions at the level of individual residues by NMR and more globally by conventional spectroscopy and solution X-ray scattering. We have compared these states with a destabilized mutant, L16A, which is predominantly denatured under conditions where the wild-type is native. This engineered denatured state, which could be directly studied under native conditions, was in genuine equilibrium with the native state, which could be observably populated by changing the conditions or introducing a stabilizing mutation. The denatured state had extensive native secondary structure and was significantly compact and globular. But, the side-chains and backbone were highly mobile. Non-cooperative melting of the residual structure on the denatured state of En-HD was observed, both at the residue and the molecular level, with increasingly denaturing conditions. The absence of a co-operative transition could result from the denatured state ensemble progressing through a series of intermediates or from a more general slide (second-order transition) from the compact form under native conditions to the more extended at highly denaturing conditions. In either case, the starting point for folding under native conditions is highly structured and already poised to adopt the native structure.
MRC Centre for Protein Engineering, Hills Road, Cambridge CB2 2QH, UK.
Ugo
U
Mayor
J Günter
JG
Grossmann
Nicholas W
NW
Foster
Stefan M V
SM
Freund
Alan R
AR
Fersht
D019943:Amino Acid Substitution; D000818:Animals; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011489:Protein Denaturation / Q000502:physiology; D013696:Temperature; D014157:Transcription Factors
D018398:Homeodomain Proteins; D014157:Transcription Factors; C106185:engrailed homeobox proteins