Tertiary stability of native and methionine-80 modified cytochrome c detected by proton-deuterium exchange using on-line Fourier transform infrared spectroscopy.
The stability of the tertiary structure of cytochrome c and of a methionine-80 chemically modified form of this protein has been investigated by monitoring on-line the exchange of amide protons with deuterons using infrared spectroscopy. The modified protein has a structural stabilization energy of approximately 50% of that of native cytochrome c, whereas the secondary structure is not affected by the modification. In the modified protein the fraction of slow exchanging amides is smaller compared to that in the native protein, and the exchange rate constants are found to be 2-3 times larger for the slow (half-life of 5.5 h) and intermediate (half-life of 4.1 min) exchanging fraction of amides. The exchange rate of a fast exchanging fraction of amides (half-life smaller than 1 min), most likely surface exposed amides, is not influenced by tertiary destabilization of the protein. The results in aqueous solution agree well with data obtained by monitoring the amide-proton exchange using 1H-nuclear magnetic resonance. In films, using attenuated total reflection infrared techniques, this difference in tertiary stability between modified and native cytochrome c could also be demonstrated. The various advantages and complications of this approach are discussed in detail.
de Jongh
H H
HH
Laboratoire de Chimie-Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, Belgium.
Goormaghtigh
E
E
Ruysschaert
J M
JM
D000577:Amides; D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c; D003903:Deuterium; D006736:Horses; D009682:Magnetic Resonance Spectroscopy; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013816:Thermodynamics
D000577:Amides; D003574:Cytochrome c Group; D011522:Protons; C091542:S-(carboxymethyl)methionine cytochrome C; D045304:Cytochromes c; D003903:Deuterium