(17) The former kinetic study further revealed that apoMb's kinetic intermediate is composed of helices A, B, G and H, and the latter study indicated that the radius of gyration is 23 A in the intermediate compared to 34 A in the unfolded state.
Therefore, the studies on the equilibrium intermediate are widely performed as a model structure for the kinetic intermediate. In this review, the strategy of a pH-pulse labeling and NMR combination method was focused on for the analysis of the kinetic intermediate of apoMb.
in this review, the analysis of the kinetic intermediate of apoMb by NMR and quick HD exchange, which is also called the pH-pulse labeling, is summarized, mainly focusing on the latter-phase kinetic (I2) intermediate (Equation , where U = unfolded protein, I1 and I2 = former-phase and latter-phase intermediates, respectively, and N = folded protein at the native state).
Analysis of the structure for the kinetic intermediate and its improved methodology
It was shown that the structure of the kinetic intermediate (probably I2 intermediate) was different between the two proteins; the structure in apoLb was composed of helices G and H, and a part of helix E (Fig.
The kinetic intermediate (probably I2) for apoLb was composed of helices G and H and a part of helix E.
Abbreviations: I2 intermediate: latter-phase kinetic intermediate; I1 intermediate: former-phase kinetic intermediate; CD: circular dichroism; apoMb: apomyoglobin; apoLb: apoleghemoglobin a; holoMb: holomyoglobin; WT: wild-type; HD exchange: hydrogen deuterium exchange; DMSO: dimethyl sulfoxide; FRET: Forster resonance energy transfer; AEDANS: 5-[[2-[(acetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid; AABUF: average area buried upon folding; HSQC: heteronuclear single quantum coherence.
(A) The proton occupancy of the kinetic intermediate (I2) and (B) the protection factor (protected ratio normalized by the intrinsic exchange rate for HD exchange) for the equilibrium intermediate of apoMb.
(A) The kinetic intermediate for apoLb and (B) the kinetic intermediate I2 for apoMb are indicated with high (red sphere), medium (green sphere), and low protections (blue sphere).
(23) Subsequently, two kinetic intermediates for both horse and whale apoMbs were demonstrated as sequential I1 and I2 by the refolding/unfolding studies monitored with fluorescence and CD including double-jump experiments.