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

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The manuscript by Konold et al describes a comparison of the photocycle kinetics of Photoactive 
Yellow Protein (PYP) measured on solution samples and crystals, respectively. PYP is a popular 
model system that has been studied using numerous approaches, including time-resolved 
crystallography. It is well known that the dynamics and kinetics of crystalline molecules can differ 
from those in solution. Crystal contacts and other steric constraints can affect mobility; the 
composition (pH, ionic strength, …) of the crystal containing solution can affect chemical reactivity. 
Thus, previous studies exist that compare the PYP photocycle in solutions and crystals. Yeremeko 
et al (Biophys J, 2006) performed studies with microsecond time resolution and noticed distinct 
differences in both time constants and pathways. Importantly, the two samples were measured at 
the same pH; the photocycle of PYP is highly pH sensitive (Hendriks & Hellingwerf, JBC 2009). 
Recently, the photocycles of PYP crystals and solutions were compared on the ultrafast time-scale 
(Hutchison et al Chem Phys Lett, 2016). Again, differences were noted, but the focus was not a 
comparison of dynamics/kinetics but to determine the parameters maximizing photocycle yield of 
the long lived pB intermediate, and by extension of intermediates occurring on the ultrafast time 
scale. An important variable was the photoexcitation power and wavelength. 
The manuscript by Konold et al compares the photocycle of crystalline and PYP using low power 
excitation, from sub-ps to seconds. The major finding is that large differences are observed on all 
time scales. Unfortunately, the crystal and solution experiments were performed at very different 
pH values (pH 8 solution, pH 6.5 crystals). It is therefore unclear whether the observed difference 
are caused by the pH, the lattice effects, or both. The authors ignore the pH effect and specify that 
the differences are due to lower hydration in the crystal, without giving any justification/proof for 
this statement (which is at most a hypothesis). Dehydration in films almost certainly differs from 
those in crystals. 
Thus, there are three major issues with this manuscript: 
1) The biggest: two important variables were changed in the experiments (pH, sample state 
(crystal/solution)), but only the latter was discussed in causing the differences. 
2) The bottom line: “Time-resolved crystallography cannot be blindly extrapolated to in vivo 
function”. This is never the case for crystal structures and well known. 
3) The differences in kinetics between solution and crystals are attributed to differences in 
hydration without offering any proof for this. 

Minor points: 
Page 3, line 2: wrong references. 
Page 3 “earliest time-resolved (and static) crystallographic: (and static) is wrong. 
Page 4 “as observed in the majority of the” .. which ones are not? 
Page 14: temperature for spectroscopic measurements? 
Page 15: Suspended drop -> hanging drop 
Page 15: crystal size? Spacer thickness? Approach similar to the one described by Hutchison et al 
Chem Phys Lett, 2016? OD of crystalline sample? What about scattering contribution? 
Laser spot diameter of 250 um: FWHM? 1/e^2? 


Reviewer #2 (Remarks to the Author): 

Konold et al. studied photocycle of Photoactive Yellow Protein (PYP) in crystal and solution by time-
resolved UV-Vis and infrared absorption spectroscopy, and compared its photocycles in a very wide 
time range from 100 femtosecond to second. On the basis of species-associated –difference-
spectra obtained with a target model, they concluded that the photocycle kinetics and the 
properties of the intermediates in the crystal significantly deviate from those in solution, and 
argued that the results of femtosecond time-resolved crystallography (TRC) cannot be blindly 
extrapolated to consider the protein in vivo. 

TRC is certainly a extremely powerful tool to study ultrafast structural dynamics of photoreceptor 
proteins and hence attracts huge attention these days. Recently, however, people are aware of 
several serious problems of the TRC experiments such as the unwanted effect induced by the 
violent excitation condition. In this paper, the authors discussed the effect of confinement in 
crystal lattice and lower hydration environment of the sample used for TRC. Because they are 
inevitable in TRC experiments, I think that the authors address a very important problem. 
Nevertheless, although I feel that the essential points of their arguments would be true, this paper 
has a number of serious problems as I write below. Therefore, I do not recommend publication of 
this paper, not only in Nature Communication but also in any other journals. 

1. A very serious problem of this paper is that the authors made most of their discussions based 
on the species-difference-spectra (SADS). SADS are very dependent on the model used in the 
analysis, and they can readily be changed with the change of the model. The authors used a very 
complicated model (given only in SI). This scheme has already been published but I do not think 
that this model is widely accepted in the community. It is fine to show and discuss SADS in the 
paper (with sufficient explanation). However, the essential discussions should be made for the raw 
time-resolved spectra which they do not show in the paper. This is critically important for readers 
to figure out which arguments are based on the time-resolved data themselves and which 
interpretations are model-dependent. Furthermore, because the author did not describe the details 
of their analysis even in Supporting Information, it is not clear which parameters they obtained 
with fitting and which values they used as fixed values. In fact, the authors discussed the 
difference in the time constant of the dynamics between crystalline-phase and solution phase, but 
I cannot follow how they reached this conclusion in a logical way. In addition, I believe that the 
authors also need to show the steady-state UV-visible and infrared spectra of PYP. 

2. One of the main arguments in this paper is that the structure of the I0 intermediate (in 
solution) is different from pR0 intermediate (in crystal), and that the I0 intermediate has the fully 
isomerized chromophore. This argument is based on the CO stretch frequency of the chromophore 
that was evaluated for time-resolved IR absorption SADS. The authors are not aware but a recent 
femtosecond impulsive stimulated Raman (TR-ISRS) study of PYP in solution clearly showed that 
the CO stretch frequency of I0 is quite low, indicating that I0 state has a distorted cis-form which 
is similar to pR0 intermediate in the crystal (Nat. Chem. 2017, 9, 660). Considering that TR-ISRS 
selectively detects the vibrations of the chromophore with the resonant Raman enhancement and 
that the S/N of the reported TR-ISRS spectrum is high, the results of the TR-ISRS study are very 
trustable. 

3. Another serious problem of this paper is that the authors do not provide sufficient descriptions 
and data in the main text. Some information is given in Supplementary Information but they did 
not make sufficient efforts to write “understandable” SI. Actually, I have an impression that the 
authors wrote this paper in a too-concise way, trying to publish it in a high-impact journal. 
Unfortunately, this makes the quality of the scholarly presentation of this paper low. This is a bad 
aspect of the trend of these days. 


Reviewer #3 (Remarks to the Author): 

This paper presents the first (to my knowledge) direct and detailed comparison of the dynamics of 
a photoactive protein in both its solution and crystalline phases. This is a key objective which will 
underpin the assessment of the reliability and usefulness of the growing number of femtosecond 
time resolved protein crystallography experiments – which are most frequently applied to 
photoactive proteins. The target here, PYP, has been thoroughly studied in solution, by ultrafast 
crystallography and by QM/MM calculation. The very important observation is made that the PYP 
photocycle dynamics (and yields) are influenced by crystallization across the entire time range 
from sub-picosecond to millisecond. Consequently it must be concluded that crystallization 
modifies even small scale (chromophore localized) dynamics, which is an important conclusion. 
Significantly, the measurements of PYPC kinetics overlap quite nicely with the crystallography 
data, which is a second useful result, given current concerns about the very levels of high 
excitation used in fs crystallography. Thus this paper contains important results in an area which is 
currently one of great importance, and should be published in NCOMM. A few suggestions for 
revision follow. 

The presentation of the observations and the data analysis are too mixed. The fact that significant 
differences are observed on all time scales is more important than identifying and characterizing 
kinetic intermediates. It is unlikely that the new intermediates found in the crystalline state are 
physiologically relevant. Thus the kinetic data (e.g. figure 1, left) should be accompanied on the 
right side of the figure by the experimental time resolved spectra, rather than the model 
dependent SADS. This will better illustrate the experimental differences in all states as well as at 
all times. The wavelength resolved data are presented in the supporting information, but these 
only allow an assessment of the fit quality – the spectra themselves will give the most striking 
evidence of the effect of packing. 

The subsequent characterization of the ‘photocycles’ (current Figure 2) following kinetic analysis to 
yield the SADS should follow the presentation of the experimental data. Were the number of 
intermediates simply determined from the minimum number that yielded an acceptable fit? If so 
could the crystalline state be exhibiting a broader distribution of relaxation rates in the early 
nanosecond time range – more a relaxation continuum than a number of additional intermediates 
– or are these intermediates truly distinct? Similarly, the actual ES lifetimes are revealed in the full 
analysis (Figure S5) to be a sum of three different ES decays; are the PYPS/C differences observed 
a consequence of a different distribution among the three ES? These details of the analysis should 
be presented, although they are probably better discussed in supplementary information and 
referenced from the main text. 

One very important result was that the same kinetics for PYPS fit both the optical and IR data. 
Was the same true for PYPC? If not what were the differences? 

The use of ‘mesoscopic’ and ‘mesoscopic context’ throughout is distracting. It suggests some 
intermediate lengthscale dependent results. However, everything in both solution and crystal is 
essentially described at the single molecule level – the effect of crystallization on the kinetics is 
discussed (very reasonably) in terms of packing and hydration levels. 

Minor matters 

In the abstract, it does not seem necessary to specify ‘during catalysis’. 
The claim in the opening paragraph for sub-Angstrom spatial resolution for IR measurements is 
perhaps an exaggeration. 
When comparing the PYPS/C SADS it may be helpful to give on the figure the rate connecting 
them as well as the state label. 
Top p9, is there some structural feature that lead to the conclusion ‘more hydrophobic’ rather than 
‘less H-bonding’? 
Top p10, is ‘weakened’ better than ‘relaxed’? 
In materials and methods please give the OD of the samples used. 
In summary, this is an important paper making a novel and important contribution to the ongoing 
assessment of the reliability and significance of femtosecond crystallography, and should be 
published in NCOMM.