Cold-Denatured Ensemble of Apomyoglobin:  Implications for the Early Steps of Folding

Abstract

The dynamics of protein-refolding experiments initiated by a temperature jump depend critically on the nature of the initial cold-denatured ensemble. The cold-denatured state of equine apomyoglobin has been investigated in aqueous buffers by near- and far-UV circular dichroism, fluorescence, infrared, and NMR spectroscopies at temperatures ranging from −20 to 98 °C. Cold denaturation of apomyoglobin is well described by a cooperative transition below 3 °C and differs in many aspects from acid-induced unfolding. As a reference system, the N-terminal A-peptide fragment of equine apomyoglobin has also been studied in aqueous and trifluoroethanol solutions. The A-peptide has a low helix-forming propensity in the absence of any stabilizing tertiary interactions. The results show that cold denaturation breaks the AGH-hydrophobic interface of equine apomyoglobin. Furthermore, at least some GH-helical structure appears to be preserved at the expense of the less stable A-helix.

Observation of strange kinetics in protein folding

“…6), kinetics at T 1 Ͻ T 2 are likely to consist of a fast D 3 DЈ phase followed by slow exponential folding over the barrier, as we observe for both PGK and Ub*G. The folding should also become more exponential as T is raised to T 3 Ͼ T 2 (heat denaturation), but this regime is presently not accessible in our experiments. Secondly, cold denaturation decreases the ruggedness of the energy landscape at low T by neutralizing both native and non-native hydrophobic contacts; this, after all, is the reason for unfolding at low temperature (22,30). As a consequence, downhill folding may not continue to stretch at very low T, as observed in Fig.…”

“…We measured slow exponential folding at lower T. The discrepancy is resolved by considering the two major effects of cold denaturation on the free energy plot (Fig. 6): first, cold denaturation is cooperative (D C and F are local minima) (22); it therefore produces a double well in the free energy at some T 1 Ͻ T 2 , even if the free energy is purely downhill at T 2 . Because the barrier becomes more pronounced and moves to smaller q at lower T (Fig.…”

“…The proteins were denatured by cooling, which causes unfolding by weakening the hydrophobic interaction (22). Refolding initiated by a nanosecond temperature jump was found to be highly nonexponential over some of the temperature range and exponential over the rest.…”

Protein denaturation by urea: Slash and bond

“…For example, important approaches to the study of protein folding dynamics are initiated from states obtained by various denaturing conditions (19). In what way are these initial states potentially different?…”

Ultrafast Peptide and Protein Dynamics by Vibrational Spectroscopy

“…Since folded and unfolded state coexist in equilibrium, important conclusion about the folding dynamics can been drawn from the unfolding dynamics. In case of cold-denatured proteins, temperature jumps may also be applied to investigate folding directly [81].…”