Peer review of: Confinement in crystal lattice alters entire photocycle pathway of the Photoactive Yellow Protein (2020)


RR2
REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author):
  The revised manuscript by Konold et al has undergone substantial changes, is much improved and
 most of my concerns have been addressed. However, the authors should focus more on what is
 new. It is well known, or at least published, that the photo cycle differs in solution and crystals. In
 the latter there are additional states such as pR1 (see e.g. Jung et al) , and the slower time scales
 deviate quite a bit due to crystal packing constraints.
 I realize that the photocycle of PYP is complex and that the different nomenclatures do not help.
 However, given the focus of the manuscript, differences on the ultrafast time scale in
 solution/crystal, I feel that it would be more appropriate to show Supplementary Fig. S6 in the
 main text than the strongly simplified Fig. 2. Even more so as the intricacies of Fig. S6 are
 described in the main text. It seems to me that the schemes shown in Fig. S6 for solution and
 crystal are rather similar? If not this needs to be stressed further and more explicitly than
 currently done. It is a pity that the authors have not made more use of the large amount of space
 available in Nature Comm.
 Along these lines, I find the manuscript rather difficult to read. It is not always clear which sample
 is being described (crystalline/solution). I understand that the authors want to directly compare
 features between the two samples instead of describing one sample first, then the other, then the
 differences but this comes at a price. Not sure it is worth is.
 I think a stringent comparison of the ultrafast photocycle in solution and crystals would be
 interesting. It is important that comparable parameters are used for the experiments. It is
 therefore very good that the authors also pointed out the reference describing the lack of influence
 of increasing the PEG concentration on the photocycle kinetics. This limits the reasons for the
 apparent discrepancy.
 There is one further difference that the authors did not mention: The orientation of protein
 embedded chromophores in solution and crystals differ, they are randomly distributed with respect
 to the photoexcitation polarization plane in solution whereas photo selection rules likely apply in
 oriented crystals (see e.g. page 19260 PYP photoactivation; Schotte et al, PNAS). The crystalline
 pancake approach, making a sample for spectroscopy by squishing a very large crystal between
 glass plates separated by thin spacers may not be good enough to randomize the orientations of
 the microcrystalline fragments. This could be avoided by stirring up the squished crystal and
 letting the crystallites settle. I could understand if the authors did not want to repeat some of the
 measurements using such a sample, but they should address this problem in the manuscript
 thoroughly. Or add a disclaimer that this could influence the kinetics in the crystalline state. I take
 it they use linearly polarized light? (not stated). SFX experiments suffer less from this issue
 because they use randomly oriented crystals for data collection and most often circularly polarized
 light.
 The authors are likely aware that there are a number of controversies associated with the time-
 resolved crystallographic data. By no means do Schotte et al and Jung et al agree, see controversy
 published in Nature Chemistry 2014, this concerns the conformation of the chromophore and the
 lack of observation of pR1 by Schotte et al. In particular the ultrafast TR-SRX experiments on PYP
 performed by Schmidt and coworkers used much higher power densities for photoexcitation than
 considered safe by Hutchinson et al., these experiments were all performed using multiphoton
 excitation. Photoexciation was mostly performed at 450 nm, the PYP crystals were grown from salt
 and not PEG. I am therefore surprised by the statement that �the PYPc results closely follow those
 by Schotte, Jung and Tenboer [misspelled]. This included... and the 22 % overall pR quantum
 yield... � Line 175-178. The quantum yield in PYP is wavelength dependent, why would it be the
 same?
 Line 244: �Changes in the region of the ... �this could be checked in the various crystal structures

 (e.g. see Table 1, Pande et al), with the caveats mentioned above
 Line 250: �Schotte et al reported that in pB, Arg52 switches to an open position..� This is correct.
 But Arg52 moves because it would otherwise directly overlap with the oxygen of the cofactor in
 the new pCa conformation in pB. The pCa oxygen is 2.9 Ang from a water molecule (2115) and 2.9
 Ang from NE of Arg52. Glu46 forms a hydrogen bond to a water molecule(2056) located at the
 position of the oxygen atom of dark state pCA (3.2 Ang). The water also forms a hydrogen bond to
 Tyr42. (Glu-O-pCa: dark2.5 Ang, pR0: 2.7 Ang, pR2: 2.6 Ang); structures from Schotte.
 Line 259: � We conclude that our transient measurements on PYPC are remarkably consistent with
 the photocycle elements as observed in the TRC experiment�. First, see caveats mentioned above.
 Second, Pande et al observes full isomerization within 0.6 ps, no further changes take place on the
 ps time scale. This contradicts the findings described Line 162-165 and 268-270. As mentioned
 above, there may be many reasons for this discrepancy. But lumping all time-resolved
 crystallographic experiments together and saying that they agree with the findings of the authors
 is not adequate for the purpose of this manuscript.
 The authors attribute dehydration as the reason for the discrepancies between PYP photocycle
 kinetics in solution and the crystalline state. Increasing PEG concentrations is used frequently by
 crystallographers to dehydrate crystals to improve packing. Doesn�t the lack of influence of
 increasing the PEG concentration on the dynamics argue against this? Yeremenko only studied
 micro to millisecond spectroscopy. In this time range the photocycle differs a lot between solution
 and crystal anyway. It does not necessarily show that PEG does not influence the kinetics on the
 fast time scale. This caveat needs to be mentioned. A nice recent example is given by Woodhouse
 Nature Comm 2020, showing that the presence of ammonium sulfate (used for crystallization) can
 change the kinetics in a fluorescent protein. Jasper van Thor thinks it is the viscosity of the protein
 that influences, or at least can influence the ultrafast dynamics upon photoexciation. He mentions
 this in one of his publications.
 The authors conclude �Overall, these results lead us to suggest that results from TRC experiments
 should be supplemented with spectroscopic experiments on proteins in solutions and crystalline
 state....� Line 297-299. As I mentioned in my first review, this is not a new idea and the authors
 are not the first to do so. The examples especially on fluorescent proteins should be cited, this
 would also be a good place for the bacteriorhodopsin reference (Line 53-55).
 In conclusion, the manuscript is much improved but still too superficial. It is a pity. The authors
 should try harder to focus on what is new.
 Minor points:
 Line 53-55: The sentence about bacteriorhodopsin is out of place here and should be moved
 elsewhere.
 Line 93: IRF? Not introduced
 Figure 1 A,B. It would be very helpful if the spectra were plotted using the same scale on the x-
 axis (wavelength) to facilitate direct comparison of the spectra. What is the reason that the
 isosbestic point differs so much?
 Line 129: to be highest for? [instead of from]
 Reviewer #2 (Remarks to the Author):
1. This paper presented elaborate comparison between PYP dynamics in solution (PYPs) and crystal
 The authors rewrote the manuscript, and the paper has become readable. This change made their
 results and arguments clearer but also disclosed the weakness of this paper. I think that this paper
 is publishable in a specialized journal but does not merit publication in Nat. Comm. I mention the
 weakness of this paper in the following.
 
 (PYPc) in a very wide time range from picosecond to millisecond. Time-absorption spectroscopy
 shows �rough� similarity between the dynamics of PYPs and PYPc, but details are noticeable
 different, as expected. This a nice demonstration but they failed to clarify the origin of the
 difference. They showed one example about a possible structural difference of the first ground
 state I0 state with time-resolved IR spectrum of the 13C analogue. Nevertheless, I0 itself is not
 the main focus of this paper, even if it is an element, so that it cannot make this paper worth
 publishing in Nat. Comm. I completely agree to the message of this paper, �even ultrafast TRC
 results cannot uncritically extrapolated to in vivo function�, but I believe that this has already
 become a consensus in the community.
 2. Although I have already mentioned it in my comments for the original manuscript, I repeat
 saying that it is necessity to show the original time-resolved infrared absorption spectra in this
 paper. I know well the usefulness of SADS but the data before any analysis are the most
 important. Actually, the authors show both original time-resolved spectra and SADS for time-
 resolved UV-visible absorption. Why don�t they do the same for time-resolved infrared data?
 Seeing the temporal traces given in SI, I start suspecting that the authors are not willing to show
 them because of the low S/N. If this is the case, it is not acceptable.
 3. In addition to the above-mentioned problems, the scholarly presentation of this paper has many
 problems. The authors might say that it is nothing to do with scientific quality but the scholarly
 presentation is also crucial if they wish to publish their work in a premier journal. I only list a few
 examples in the following: the figure number is different from the order of the appearance (Figures
 2 and 3); Combination of the four figures in Figure 2 does not well match the descriptions in the
 main text; Both of original time-resolved spectra and SADS are given for transient UV-visible
 absorption but only SADS is given for transient infrared absorption; a fatal typo about figure
 number, i.e., Figure 2B,C reads Figure2C,D in Line141; inconsistent number about the vibrational
 frequency between the main text and figure, i.e., 1685 cm-1 (Line104) vs. 1684 cm-1 (Figure 3).
 Reviewer #3 (Remarks to the Author):
 I have read the response to reviewers and the revised paper. From my (reviewer 3) point of view
 the main matters have been addressed. With reviewer 2 I am pleased to see that actual data has
 been included, and forms the basis for the initial mechanistic discussion. This adds certainty to the
 main conclusion that TRC and TR solutions experiments can provide different dynamics. As TRC
 becomes more prevalent realizing this will become more pressing.
 I see from other comments that there are shades of opinion on assignment of structures to
 intermediates in PYP. I am not competent to assess such specialist points for PYP.
 In my view the paper can be published, and is an important contribution to field of interpreting
 TRC data.