Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

Abstract

In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.

Folding of apomyoglobin: Analysis of transient intermediate structure during refolding using quick hydrogen deuterium exchange and NMR

“…Furthermore, the continuous-flow mixer was employed for the studies on the early stage of folding of whale and horse apoMbs. 37 , 38 , 58 ) More than two intermediates were identified for apoMb folding based on the refolding/unfolding studies of urea-jump at acidic and neutral pHs, monitored by the fluorescence and CD. Laser-induced oxidative labeling after the initiation of folding by rapid mixing was combined with the mass spectrometry-based peptide mapping for the studies on the folding of horse apoMb.…”

“…It was also recently shown that the former-phase kinetic intermediate for apoMb (I1) contains one more intermediate. 37 , 38 ) However, for the purpose of simplifying considerations of this folding system, the I1 intermediate is treated as representing a single structural species in this review.…”

Proteins Take up Water Before Unfolding

“…Previous studies showed that for many proteins, equilibrium unfolding intermediates are identical to kinetic unfolding intermediates in terms of their secondary and tertiary structure content, hydrogen exchange protection, collision cross-section, and stability toward unfolding. − Hence, mild denaturation conditions can be associated with earlier stages of unfolding. This means that our observation that proteins become more accessible to water at low urea concentrations, indicates that unfolding starts with the protein becoming less tight, allowing water to enter.…”

Statistical Mechanical Model for pH-Induced Protein Folding: Application to Apomyoglobin

“…On the basis of the above considerations and previous studies on pH-induced folding of h- and sw-apoMb, ,, we assumed a five-state sequential model (Scheme ), which is also fully consistent with urea-induced refolding/unfolding at pH 6.0, to reproduce the pH-induced refolding/unfolding kinetics of h-apoMb. The need for five states was also confirmed in terms of the rate equation.…”

“…In addition, the Gibbs free energy difference between two states was determined by the ratio of the partition functions, which corresponded to the difference in the potential energies (represented as the exponents of the partition functions), which required a set of constrained parameters in a reference state (U in this study). Reference elementary rate constants ( k 0 kl ), which were equivalent to the reference activation free energies (eq ), and the fluorescence intensity of each species at pH 6.0 were obtained from a previous report on urea-induced refolding/unfolding of h-apoMb at 0 M urea and pH 6.0 . The reference elementary rate constants at pH 6.0 were constrained as fixed parameters to define the reference free energy of each state.…”