Comment on mr-2021-16

In section 2, apart from discussions on thermodynamic and kinetic information to be gained, the review goes directly into novel experimental methodologies with hardware development and pulse sequences. Particularly, the methodologies for inducing nonequilibrium conditions in target macromolecular systems and the associated rf pulse sequences for NMR observation. Representative hardware setups for triggering nonequilibrium conditions are shown by photos, and the strategic rf pulse sequences for observing time-dependent NMR signals are given in another figure (Figure 5 along with Table 1). For time-resolved irreversible experiments, which do not allow signal accumulations, a few alternative methods for increasing effective NMR sensitivity are introduced in section 3. In section 4, various time-resolved NMR spectroscopies are used to follow various protein dynamics including

In section 2, apart from discussions on thermodynamic and kinetic information to be gained, the review goes directly into novel experimental methodologies with hardware development and pulse sequences. Particularly, the methodologies for inducing nonequilibrium conditions in target macromolecular systems and the associated rf pulse sequences for NMR observation. Representative hardware setups for triggering nonequilibrium conditions are shown by photos, and the strategic rf pulse sequences for observing time-dependent NMR signals are given in another figure (Figure 5 along with  Table 1). For time-resolved irreversible experiments, which do not allow signal accumulations, a few alternative methods for increasing effective NMR sensitivity are introduced in section 3. In section 4, various time-resolved NMR spectroscopies are used to follow various protein dynamics including light and rapid mixing applications. photo-CIDNP for studying dynamics in proteins. The method has been extended to include nucleic acids.
In summary, the review is an excellent contribution regarding the major topics and advancement in recent years pertaining to the title issue, giving an overall view of the frontier field of NMR spectroscopy and biopolymer science.
Unfortunately, there appears some lack of recognition by the authors on the historical background in some area of science, which I would like to put some comments on, in addition to several minor errors which I'd like to be corrected.

Questions and suggestions
Question 1.
Are the folding intermediates detectable only by the slow time-dependent experiment, i.e., by real-time NMR ?
The authors' comment below (in Page 3, lines 86-89) seems to support this: "While equilibrium studies focus on characterization of conformational transitions in the microsecond-to-millisecond time scale involving NOESY-type experiments (Evans et al., 1989), line shape analysis (Evans et al., 1989;Huang and Oas, 1995) or relaxation dispersion (Korzhnev et al., 2004), non-equilibrium studies focus on slower biomolecular folding transitions." (By this comment, the authors seem to be telling that the information on the folding intermediates may be obtained only from slow time-dependent experiments, namely, the real-time NMR spectroscopy.) This statement does not apply to the high-pressure NMR experiment of proteins performed under static pressures. Indeed, we have practically overcome this limitation already in 2003, by utilizing the high-pressure NMR spectroscopy carried out under elevated, but static pressures; Indeed, we found; 1) At certain levels of pressure, we detect signals from "intermediate" conformers.
In general, with the high-pressure NMR spectroscopy carried out under static hydrostatic pressures, we can identify intermediates as distinctly different thermodynamic entity from that of the basic folded conformer, and determine their structures stably trapped under pressure in fair detail (cf. Akasaka, K., Kitahara, R., Kamatari, Y. O, (2003), in Advances in High Pressure Bioscience and Biotechnology II (R. Winter, Ed.) pp. 9-14; Akasaka, K. (2018), Protein Studies by High Pressure NMR. in Experimental Approaches of NMR Spectroscopy (Chapter 1), pp. Springer). Under the circumstance, combining the high-level structural information along with thermodynamic information obtainable from the "static" high-pressure NMR experiment (Akasaka, K. (2003). Pure Appl. Chem., 75, 927-936.; Akasaka, K. (2003). Biochemistry, 42(37), 10875-10885); Akasaka, K. (2006). Chemical Reviews, 106, 1814-1835) with the information obtainable from the kinetic information from real-time NMR spectroscopy as exemplified in this review seems to me to a promising way toward better understanding of protein folding studies.

Question 2.
Proper reference citation requested for the "State-Correlated 2D NMR Spectroscopy" In Page 11 (both in the text and in Figure legend (Fig. 3)), the term "State-Correlated (SC)" spectroscopy is mentioned, Here, no reference is made to, the original paper in which the first "State-Correlated (SC)" spectroscopy was carried out with its naming (the paper (Naito, Nakatani, Imanari and Akasaka, J. Magn. Reson. 1990, 87 429-432)).
Early history of State-Correlated 2D NMR Spectroscopy: In 1989, our idea was to demonstrate a new type of two-dimensional NMR spectroscopy using our newly developed microwave T-jump NMR apparatus, which would correlate the states of spin magnetization of a molecule embedded in two different thermodynamic states. In 1990, the first such experiment was performed to correlate the signals of water protons at two different thermodynamic states (in fact, two different temperatures), which was performed with the home-made microwave T jump apparatus with an innovative design employing the microwave heating assisted by a dielectric resonator (cf. Kawakami, M., & Akasaka, K. (1998). Microwave temperature-jump nuclear magnetic resonance system for aqueous solutions. Review of Scientific Instruments, 69(9). https://doi.org/10.1063/1.1149102), We were successful in obtaining the first twodimensional spectrum of water protons connecting the two temperatures and published the result with the title "State-Correlated (SC) two-dimensional NMR spectroscopy" (Naito, Nakatani, Imanari and Akasaka, J. Magn. Reson. 1990, 87 429-432). One year later, in 1991, the first SC-2D experiment was performed on a protein in solution (cf. Akasaka, K. et al., Novel method for NMR spectral correlation between the native state conformer and the heat-excited state (probably closely heat denatured state) of protein ribonuclease A. J. Am. Chem. Soc. 1991, 113, 4688). Later, the method was extended to separate signals in liquid crystals (cf. Naito et al., J. Chem. Phys.1996,105, 4504).
Proper reference citation requested: I would appreciate the authors to cite this original and classical article on the SC-2D spectroscopy, Naito, Nakatani, Imanari and Akasaka, J. Magn. Reson. 1990, 87 429-432, in the text (line 243) and in the legend of Figure 5 in page 11. Is it possible to revise the figure in Fig. 5 for SC-2D experiment to be remade in the direction to cope with the standard 2D NMR spectroscopy (by Richard Ernst) like that in Fig. 1, Akasaka et al. (1991). J. Am. Chem. Soc. 113, 4688-4689?

Corrections/Additions
Page 4; lines 100-105; The general statement here on the effect of pressure-jump and temperature-jump on biomolecular folding and refolding appears inappropriate. The effect of pressure on protein conformational stability is primarily determined by the volume change of the system, while the effect of temperature on the conformational stability is primarily determined through the heat capacity change of hydration.
Page 5: lines 137-145; The effect of pressure on bio-macromolecular structure in aqueous solution should better be discussed on thermodynamic terms. The literatures cited here are not considered appropriate. I urge the authors to reconsider the citations here to include the more fundamental ones.