Proton resonance assignments of horse ferricytochrome c

Abstract

Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) was used to obtain extensive resonance assignments in the 1H NMR spectrum of horse ferricytochrome c. Assignments were made for the main-chain and C beta protons of 102 residues (all except Pro-44 and Gly-84) and the majority of side-chain protons. As starting points for the assignment of the oxidized protein, a limited set of protons was initially assigned by use of 2D NMR magnetization transfer methods to correlate resonances in the oxidized form with assigned resonances in the reduced form [Wand, A. J., Di Stefano, D. L., Feng, Y., Roder, H., & Englander, S. W. (1989) Biochemistry (preceding paper in this issue)]. Given the complexity of the spectrum due to the size of this protein (104 residues) and its paramagnetic center, the initial search for side-chain spin systems in J-correlated spectra was successful only for the simplest side chains, but the majority of NH-C alpha H-C beta H subspin systems (NAB sets) could be identified at this stage. The subsequent search for sequential NOE connectivities focused on NAB sets, with use of previously assigned residues to place NOE-connected segments within the amino acid sequence. Selective proton labeling of either the slowly or the rapidly exchanging amide sites was used to simplify the spectra, and systematic work at two temperatures was used to resolve ambiguities in the 2D NMR spectra. These approaches, together with the use of magnetization transfer methods to correlate reduced and oxidized cytochrome c spectra, provide multiple cross-checks to verify assignments.

Monitoring the conformational flexibility of cytochrome c at low ionic strength by 1H‐NMR spectroscopy

“…The major and minor forms (A and B, respectively) are in slow exchange with respect to the separation in chemical shift. The A form has 1 H-NMR signals with the same shifts as in 100 mM sodium phosphate solutions [49,50]. The same behavior in the 1 H-NMR spectra is observed when cyt c is dissolved in 1 mM sodium phosphate (where a pH Ϸ9 is obtained and the alkaline form is present), then the pH is adjusted by addition of phosphoric acid down to pH 7.…”

13C and proton NMR studies of horse cytochrome c

“…Most 3C chemical shifts were specifically assigned by means of two-dimensional shift correlation experiments [2] using existing proton assignments [8,9]. The proton decoupler provided field strengths corresponding to 90 O pulses of 34 ps when used for polarisation transfer and of 110 ps for decoupling with 10-mm diameter sample tubes.…”

The Conformational Flexibility of Oxidized Cytochrome c Studied through Its Interaction with NH3 and at High Temperatures

“…At 293 K only the resonances of the native form are detected [10] [54] . Upon increasing the temperature, new hyperfine shifted resonances appear in the 25Ϫ20 and 10Ϫ13 ppm ranges; a broad signal is also observed at δ ϭ Ϫ10.…”