Localising individual atoms of tryptophan side chains in the metallo-<i>β</i>-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites

Orton, Henry W.; Herath, Iresha D.; Maleckis, Ansis; Jabar, Shereen; Szabo, Monika; Graham, Bim; Breen, Colum; Topping, Lydia; Butler, Stephen J.; Otting, Gottfried

doi:https://doi.org/10.5194/mr-3-1-2022

Articles | Volume 3, issue 1

https://doi.org/10.5194/mr-3-1-2022

© Author(s) 2022. This work is distributed under
the Creative Commons Attribution 4.0 License.

https://doi.org/10.5194/mr-3-1-2022

© Author(s) 2022. This work is distributed under
the Creative Commons Attribution 4.0 License.

Articles | Volume 3, issue 1

Research article

|

04 Jan 2022

Research article |

| 04 Jan 2022

Localising individual atoms of tryptophan side chains in the metallo-β-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites

Henry W. Orton, Iresha D. Herath, Ansis Maleckis, Shereen Jabar, Monika Szabo, Bim Graham, Colum Breen, Lydia Topping, Stephen J. Butler, and Gottfried Otting

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Interactive discussion

Status: closed

RC1: 'Comment on mr-2021-63', Marcellus Ubbink, 15 Oct 2021

This manuscript describes how pseudocontact shifts generated with a lanthanoid tag attached to three different positions on a protein can be used to determine the position of a Trp indole ring in a protein. The position of the indole of Trp 28 in the metallo-beta-lactamase IMP-1 is a matter of debate because crystal structures give different conformations. The conformation was determined in the resting state and with an inhibitor bound and turned out to be very similar. The experimental work appears sounds and the analysis is supported by the data. The authors also discuss problems encountered, which is very useful. The results are highly relevant for a better understanding of this enzyme, so the work should be published.

Some points need addressing.

(1) The triangulation approach of using the intersections of three PCS isotherms has been reported before in other papers (e.g. J Biomol NMR 71, 27, 2018), so it is not clear why the current approach is not compared with published methods.

(2) In the discussion (l. 479 – 486), the point of not using different metals in the same tag but multiple orthogonal sites has been made by other studies, so references are required there.

(3) In line 246 it is mentioned that double peaks are observed for the Trp NHe groups. That could mean that the indoles are in different conformations in slow exchange. It is not discussed whether these could be the other conformations observed in the crystal structures. Could the PCS analysis be done for these minor peaks to exclude that possibility? In that case this work only yields the position of the major form, not the only form.

(4) The mass spectrometry shown in Fig. S2 and the yields mentioned give rise to questions. In line 204 the efficiency of 90% is mentioned. However, using the information in the caption of Fig. S2, a different result is suggested: The masses in the figure are about 9 Da lower than expected for 100% labelling (given the mentioned masses), which 25% of the expected 36 Da extra (in the caption it says +6 Da/Trp), so labelling efficiency would be 75%. However, after converting the indole to tryptophan, one deuteron is removed, so the expected mass increase is 4XD + 1 13C = 5 Da per Trp, not 6. That would result in a labelling of 90%, agreeing with the main text, but suggesting that the masses mentioned in the caption are too high. Please check.

(5) In section 2.2, mention the protein concentration(s) used for the NMR samples and indicate the tube type (3 mm, 5 mm, Shigemi), to know how much sample was used. Also mention the protein concentration in the captions of the NMR spectra figures in the supplementary material.

(6) l. 213, how were the assignments for the diamagnetic protein obtained? If from previous reports, give the reference and the BMRB entry.

(7) Some supplementary figures have the wrong numbers in the text:

line 165, Fig S1 > S8;

line 170, Fig. S2 > S9;

line 212, Fig. S2-S5, S3-S6 (?)

Tables S1 – S6 are not mentioned in the text.

Citation: https://doi.org/10.5194/mr-2021-63-RC1
AC1: 'Comment on mr-2021-63', Gottfried Otting, 23 Oct 2021

See the uploaded supplement for an author comment with formatting.

Citation: https://doi.org/10.5194/mr-2021-63-AC1
RC2:
'Comment on mr-2021-63', Daniel Häussinger, 29 Oct 2021

The manuscript by H. Orton et al. describes the localisation of tryptophane side chain protons by pseudocontact shifts generated by two lanthanoid tags at three different attachment sites in IMP-1. The contribution is scientifically sound, highly relevant for biological applications and demonstrates elegant and novel state-of-the-art techniques to obtain suitable selectively isotope labelled protein constructs for efficient PCS triangulation. The well-balanced conclusions drawn on the conformation of the L3-loop of IMP-1 are fully supported by the presented data. In my view this is a very suitable contribution to Magnetic Resonance and I recommend the publication after a few minor revisions:

Points to be addressed:

1) The procedure of the triangulation by PCS is not new and earlier work by some of the authors and others should be referenced accordingly.

2) The finding, that two sets of PCS created at the same tagging site and by the same tag, but different lanthanoids give less accurate results, is not new but certainly remarkable – it would be tempting to quantify this finding by elucidating the “angle score” parameter for these data sets as suggested by Joss et al. (, 2019, , 5064-5072.)

3) The authors refer only to proton PCS despite the fact, that they obtained also PCS data from ¹⁵N and ¹³C – could you comment on that?

4) The description of the MS instrumentation and conditions is missing.

5) The suggested incorporation level of the ¹³C-indole is not in accordance with the presented MS spectra in Fig S2, panels a-c.

The figure should be reproduced with better resolution of the individual spectra to allow judgement of the incorporation and a deconvolution of the different isotopomers should be provided.

6) It would be useful to have the individual metal – cysteine-sulphur distances for each tensor included in table S7

7) In the experimental section it is reported that the conjugation of the tags to the protein was performed in the presence of 100 uM ZnSO₄ (buffer A), does this not interfere with the cysteines, given the nM Kd values of Zn(Cysteine)₂ complexes?

8) minor typos:

line 142: should read “preparation of each sample”

line 165 should read “Fig. S8”

line 170 should read “Fig. S9”

line 204 c.f. 5)

line 207: “100%” on a deconvoluted mass spectrum is probably stretching the significance a bit – how about “virtually quantitative” or “ > 95%”?

line 346: the “of” in “isosurfaces of associated” seems superfluous to me.

Citation: https://doi.org/10.5194/mr-2021-63-RC2
- AC2: 'Reply on RC2', Gottfried Otting, 30 Oct 2021
  
  1) The procedure of the triangulation by PCS is not new and earlier work by some of the authors and others should be referenced accordingly.
  Response: we agree and propose to include the following (broadly representative though still incomplete) list of references in the introduction (this is the same response as to comment 1 by Marcellus Ubbink):
  For side chain conformations:
  Pearce, B. J. G. Jabar, S., Loh, C.-T., Szabo, M., Graham, B. and Otting, G.: Structure restraints from heteronuclear pseudocontact shifts generated by lanthanide tags at two different sites, J. Biomol. NMR, 68, 19–32, https://doi.org/10.1007/s10858-017-0111-z, 2017.
  Lescanne, M., Ahuja, P., Blok, A., Timmer, M., Akerud, T. and Ubbink, M. Methyl group reorientation under ligand binding probed by pseudocontact shifts, J. Biomol. NMR, 71, 275–285, https://doi.org/10.1007/s10858-018-0190-5, 2018.
  For protein-ligand complexes:
  Guan, J.-Y., Keizers, P. H. J., Liu, W.-M., Löhr, F., Skinner, S. P., Heeneman, E. A., Schwalbe, H., Ubbink, M. and Siegal, G.: Small-molecule binding sites on proteins established by paramagnetic NMR spectroscopy, J. Am. Chem. Soc., 135, 5859–5868, https://doi.org/10.1021/ja401323m, 2013.
  Chen, W.-N., Nitsche, C., Pilla, K. B., Graham, B., Huber, T., Klein, C. D. and Otting, G.: Sensitive NMR approach for determining the binding mode of tightly binding ligand molecules to protein targets, J. Am. Chem. Soc., 138, 4539–4546, https://doi.org/10.1021/jacs.6b00416, 2016.
  For protein-protein complexes:
  Pintacuda, G., Park, A. Y., Keniry, M. A., Dixon, N. E. and Otting, G.: Lanthanide labeling offers fast NMR approach to 3D structure determinations of protein-protein complexes, J. Am. Chem. Soc., 128, 3696–3702, https://doi.org/10.1021/ja057008z, 2006.
  Keizers, P. H. J., Mersinli, B., Reinle, W., Donauer, J., Hiruma, Y., Hannemann, F., Overhand, M., Bernhardt, R. and Ubbink, M.: A solution model of the complex formed by adrenodoxin and adrenodoxin reductase determined by paramagnetic NMR spectroscopy, Biochemistry, 49, 6846–6855, https://doi.org/10.1021/bi100598f, 2010.
  de la Cruz, L., Nguyen, T. H. D., Ozawa, K., Shin, J., Graham, B., Huber, T. and Otting, G.: Binding of low molecular weight inhibitors promotes large conformational changes in the dengue virus NS2b-NS3 protease: fold analysis by pseudocontact shifts, J. Am. Chem. Soc., 133, 19205–19215, https://doi.org/10.1021/ja208435s, 2011.
  Kobashigawa, Y., Saio, T., Ushio, M., Sekiguchi, M., Yokochi, M., Ogura, K. and Inagaki, F.: Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein-protein complex structure determination, J. Biomol. NMR, 53, 53–63, https://doi.org/10.1007/s10858-012-9623-8, 2012.
  Brewer, K. D., Bacaj, T., Cavalli, A., Camilloni, C., Swarbrick, J. D., Liu, J., Zhou, A., Zhou, P., Barlow, N., Xu, J., Seven, A. B., Prinslow, E. A., Voleti, R., Häussinger, D., Bonvin, A. M. J. J., Tomchick, D. R., Vendruscolo, M., Graham, B., Südhof and T. C., Rizo, J.: Dynamic binding mode of a synaptotagmin-1-SNARE complex in solution. Nat. Struct. Mol. Biol., 22, 555–564, https://doi.org/10.1038/nsmb.3035, 2015.
  For 3D protein structure determination:
  Yagi, H., Pilla, K. B., Maleckis, A., Graham, B., Huber, T. and Otting, G.: Three-dimensional protein fold determination from backbone amide pseudocontact shifts generated by lanthanide tags at multiple sites, Structure, 21, 883–890, https://doi.org/10.1016/j.str.2013.04.001, 2013.
  Crick, D. J., Wang, J. X., Graham, B., Swarbrick, J. D., Mott, H. R. and Nietlispach, D.: Integral membrane protein structure determination using pseudocontact shifts. J. Biomol. NMR, 61, 197–207, https://doi.org/10.1007/s10858-015-9899-6, 2015.
  Pilla, K. B., Otting, G. and Huber, T.: Protein structure determination by assembling super-secondary structure motifs using pseudocontact shifts, Structure, 25, 559–568, https://doi.org/10.1016/j.str.2017.01.011, 2017.
  
  2) The finding, that two sets of PCS created at the same tagging site and by the same tag, but different lanthanoids give less accurate results, is not new but certainly remarkable – it would be tempting to quantify this finding by elucidating the “angle score” parameter for these data sets as suggested by Joss et al. (Chem. Sci., 2019, 10, 5064-5072.)
  Response: The angle score parameter is useful. In a comprehensive comparison, we recently explored the relative merits of labelling a single site with different tags versus placing the same tag at multiple sites, considering different scores to quantify the information content of different PCS isosurfaces. The results are not yet quite complete and we therefore intend to publish them separately. We suggest the following new paragraph (which is the same as in our response to comment 2 by Marcellus Ubbink):
  It has been pointed out previously that the accuracy with which localisation spaces can be determined is best when PCS isosurfaces intersect in an orthogonal manner (Pintacuda et al., 2006; Lescanne et al., 2018; Zimmermann et al., 2021). In the present work, we found that, counterintuitively, the provision of additional data can considerably degrade the accuracy of the localisation space. This effect arises when PCS isosurfaces intersect at a shallow angle, as the location of these intersections becomes very sensitive with regard to small errors in the relative orientations of the underpinning Dc tensors. Shallow intersection angles of PCS isosurfaces routinely occur, when two PCS datasets are from tags and tagging sites that differ only in the identity of the paramagnetic metal ion in the tag. Thissituation commonly generates Dc tensors of different magnitude and sign, but closely similar orientation (Bertini et al., 2001; Su et al., 2008; Keizers et al., 2008; Man et al., 2010; Graham et al., 2011; Joss et al., 2018; Zimmermann et al., 2021). Therefore, while the use of Tm³⁺ and Tb³⁺ tags is helpful for assigning the cross-peaks in the paramagnetic state, it is safer to use only one of these data sets for calculating the localisation space. Good localisation spaces were thus obtained by using only PCSs measured for Tb³⁺ tags (Fig. 6) or only PCSs measured for Tm³⁺ tags (Fig. S12). In contrast, however, very different tags attached at the same site, such as the C2 and C12 tags installed in the mutant N172C, produced independent Dc-tensor orientations and therefore contributed positively to localising the Trp28 H^e1atom.
  
  3) The authors refer only to proton PCS despite the fact, that they obtained also PCS data from ¹⁵N and ¹³C – could you comment on that?
  Response: We use only ¹H PCSs because nuclear spins with large CSA tensors display residual anisotropic chemical shift (RACS) effects due to weak paramagnetic alignment of the molecule in the magnetic field. The mixture of alignment and PCS information is cumbersome to disentangle. Furthermore, paramagnetic shifts measured of heteronuclei in the indirect dimension tend to come with greater uncertainties. We suggest to add the following paragraph in the discussion of the revised version:
  The present work employed ¹H PCSs only, although PCSs were also observed in the indirect dimensions of the [¹³C,¹H]-HSQC and [¹⁵N,¹H]-HSQC spectra. We made this choice because the paramagnetic tags give rise to weak molecular alignments in the magnetic field, which result in residual anisotropic chemical shifts (RACS). The effect is unimportant for ¹H spins but significant for nuclear spins with large chemical shift anisotropy (CSA) tensors such as backbone nitrogens and aromatic carbons. Correcting for the RACS effect is possible with prior knowledge of the CSA tensors and bond orientations (John et al., 2005). We therefore chose not to measure PCSs of the heteronuclear spins in favour of improving sensitivity by accepting a lower spectral resolution in the indirect dimensions.
  
  4) The description of the MS instrumentation and conditions is missing.
  Response: We propose to add the following information in the supporting information:
  Protein mass spectrometry
  Intact protein analysis was carried out on an Orbitrap Elite Hybrid Ion Trap–Orbitrap mass spectrometer connected to an UltiMate 3000 UHPLC (Thermo Scientific, USA). 0.1% formic acid and an acetonitrile gradient (10–85 %) were used to inject the samples into the mass analyzer via an Agilent ZORBAX SB-C3 Rapid Resolution HT Threaded Column. Data were collected in positive ion mode using an electrospray ionization (ESI) source. The Xtract function in the Qual Browser software tool of the program Xcalibur 3.0.63 was used to deconvolute and determine the protein intact mass. (Thermo Fisher Scientific, USA).
  
  5) The suggested incorporation level of the ¹³C-indole is not in accordance with the presented MS spectra in Fig S2, panels a-c. The figure should be reproduced with better resolution of the individual spectra to allow judgement of the incorporation and a deconvolution of the different isotopomers should be provided.
  Response: Some of the data in Figure S2 were swapped by mistake. We looked at the data again carefully and concluded that they are in better agreement with an incorporation yield of the isotope-labelled indole of about 80 % (rather than 90 % as claimed in the original version of the manuscript). This conclusion comes from observing the maxima of the measured isotopomer distributions at masses between 3 and 6 Da lower than expected for 100 % incorporation. (Mutant A53C, measured: 25232.45; calculated: 25235. N172C, measured: 25189.40; calculated: 25192. S204C, measured: 25213.27; calculated: 25219. We feel that calculating the expected mass with greater precision is not meaningful, as masses measured of IMP-1 on our instrument were reproducible within not much better than one mass unit (despite better instrument specification). We are providing an expanded version of the mass peaks of the mutant A53C (Figure S2a) below.
  Extracting information about the percentage of isotope-labelled tryptophans from the width of the isotope distribution would require modelling, which we believe may not be much more accurate than what can be determined from the mass of the tallest peak in the distribution.
  
  6) It would be useful to have the individual metal – cysteine-sulphur distances for each tensor included in table S7
  Response: We will do this, but propose to report the metal-to-beta-carbon distances, as we don’t know the coordinates of the sulfur atoms. These distances vary between 8.2 and 12.1 Å.
  
  7) In the experimental section it is reported that the conjugation of the tags to the protein was performed in the presence of 100 uM ZnSO₄ (buffer A), does this not interfere with the cysteines, given the nM Kd values of Zn(Cysteine)₂ complexes?
  Response: The presence of zinc was necessary as IMP-1 binds traces of iron more tightly (Carruthers et al., Angew. Chem. Int. Ed. 2014, 53, 14268). A single sidechain thiol group doesn’t bind zinc very tightly. The solutions contained no free cysteine and there was no evidence for zinc interfering in the ligation reaction.
  
  8) minor typos:
  line 142: should read “preparation of each sample”
  line 165 should read “Fig. S8”
  line 170 should read “Fig. S9”
  line 204 c.f. 5)
  line 207: “100%” on a deconvoluted mass spectrum is probably stretching the significance a bit – how about “virtually quantitative” or “ > 95%”?
  line 346: the “of” in “isosurfaces of associated” seems superfluous to me.
  Response: We are grateful for the careful evaluation and will fix these issues in the revised version. We accept that it is rarely justified to talk of 100 % yield and propose to change to “Ligation yields with the C2 tags were practically complete”.
  
  Citation: https://doi.org/10.5194/mr-2021-63-AC2

Peer review completion

AR: Author's response | RR: Referee report | ED: Editor decision | EF: Editorial file upload

AR by Gottfried Otting on behalf of the Authors (06 Nov 2021) Author's response Author's tracked changes Manuscript

ED: Reconsider after major revisions (further review by editor and referees) (11 Nov 2021) by Stephan Grzesiek

Dear Gottfried

Thanks very much for submitting this interesting article to MR. As you have seen, the two referee comments are largely positive, but they also raise a number of minor issues.

I am not yet completely familiar with the editorial process, but I would like you to also consider the points listed below that I encountered during my own reading.

Thus I will be happy to consider a revised manuscript in which you address all points.

With best wishes
Stephan

Major:

1. The major goal of the manuscript is to define the conformation/dynamics around W28 in solution. However, this is not very much worked out. I found the description on lines 401–409 very confusing. Apparently, the PCS of Trp28 Heps1 is scaled down without captopril (Fig. S5b, line 403). Contrary to what is stated on lines 404, 405 I could not see the backbone amide PCS in Fig. S11. Why would this suggest different loop conformations for differently labeled samples (line 406)? In my understanding, the reduced PCS of Trp28 Heps1 in the absence of captopril may very well indicate flexibility. This would also agree with the observed flexibility of the L3 loop and the Trp28 sidechain in the mentioned NMR relaxation study (line 71). The entire discussion on lines 401–409 should be clarified and the comparison to the earlier relaxation data should be made. Eventually this should rather be a key point of the conclusion section.

2. The minor species in the spectra seem a major concern. This problem should be explained more carefully with respect to the origin of the degradation or minor conformations and the interpretation of the data. The description in Lines 243-251 should be expanded and the interpretation should also be taken up again in the discussion, e.g. why should these minor peaks not be subconformations that correspond to some of the X-ray observations. In this respect all the additional peaks (minor species or unassigned) should be properly labeled (asterisks etc) in all the spectra, i.e. Figure 3, S3, S5, S6, S7. In this respect ‘S5’ on lines 245, 248 should probably be ‘S6’.

Minor:
1. It is very confusing that the number notation and the IUPAC notation are used interchangeably for the isotope labeling of the Trp sidechain and of indole. My personal preference would be IUPAC. Of course, this is your decision, but please use only one consistently. In any case, it would be very helpful to show the Trp/indole chemical structure with suitable numbering in one of the main figures.

2. The labeling of all spectral figures is not very reader-friendly. It would very helpful to label the subpanels with experimental conditions such that one can immediately understand the differences. E.g. Figure 2: add mutations and experiments (NOE-relay, HSQC) to subpanel rows and columns as well as color legend. This applies to all others as well: Figure 3, S3, S4, S5, S6, S7.

3.line 283: the Q factors should be put into Table S7 and this Table should be referenced in the main text.

4. line 302: ‘[13C,1H]-HSQC spectra with 150 ms NOE’ seems a misnomer.

5. line 344: ‘Fig. 5’ is apparently ‘Fig. 6’

6. Figures S3, S4, S6: please indicate Trp sidechain assignments where available.

7. Figure S13: There is no red and blue points in the figure. They are rather green and magenta in my PDF. Also in my impression, the localization spaces indicated by these points don’t seem to be the same on left and right side.

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AR by Gottfried Otting on behalf of the Authors (13 Nov 2021) Author's response Author's tracked changes Manuscript

ED: Referee Nomination & Report Request started (19 Nov 2021) by Stephan Grzesiek

RR by Marcellus Ubbink (25 Nov 2021)

RR by Daniel Häussinger (07 Dec 2021)

RR by Nico Tjandra (08 Dec 2021)

Suggestions for revision or reasons for rejection

The manuscript by Orton et. al. has been revised in response to the previous reviewers’ comments. I believe the authors have addressed all of them sufficiently. I am however quite surprised that no experimental error was mentioned anywhere in the manuscript. The authors seem to use RMSD of PCS fit to the structure as the estimated error. Please see some potential points that the authors might want to consider:

1. I suspect that the erros in the tensor parameters are rather small since a large number of backbone PCS were used to fit the tensor to the structure s of the protein in various forms. In fact the authors did a 20% random deletion to estimate the error. Can this standard of deviation of the parameters be added to the table S7.

2. Line 436 the authors state “within the uncertainty of the experiments”. So what is the uncertainty? Note that PCS as low as 1-4 ppb are listed in Table S3-S5.
I think quantifying this is quite relevant as the authors discuss the PCS analysis for W28 He1 in N172C mutant. The PCS value for that specific proton apparently is different in the two (H-C or H-N) spectra and yet using either value doesn’t seem to alter the final position of that proton in the structure. This suggests to me that for that particular orientation and distance combination, large deviation in the PCS value doesn’t translate to large spatial change. Yet in line 487, the authors concluded that W28 indole side chain can be determined “with remarkable accuracy”. As written these sections are contradictory to me. What is the spatial accuracy?

3. The authors correctly stated on line 361 that experimental error affect the PCS isosurface in a non-isotropic way. In fact, I would argue that it is really a shell with varying thickness (due to experimental error) rather than a 2D iso-surface that should be considered due to experimental error. Choosing Ln tagging site such that the area of interest is within the smallest volume of that “iso-shell” can be very important as exemplified by comment 2 above.

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ED: Publish subject to minor revisions (review by editor) (11 Dec 2021) by Stephan Grzesiek

December 11, 2021

Dear Gottfried

Thank you very much for your patience with the reviewing process of your manuscript, which was compounded by the late declaration of a conflict of interest of one of the two initial referees.

Your revised manuscript has now been seen again by the two initial referees who are satisfied with the changes and recommend publication as is.

In addition, I had sent the manuscript to a third referee to evade the conflict of interest. The third referee also recommends publication after addressing some minor points. I think these points are interesting and addressing them will make the manuscript stronger without causing much work.

Furthermore you had also replied to my suggestions for improvements, which I feel have been addressed to a large extent. There is one remaining minor point in your answer:

"6. Figures S3, S4, S6: please indicate Trp sidechain assignments where available. Response:
Figs S3, S4 and S7 in ms_current serve as overviews of the 15N-HSQC spectra. To assign all the Trp sidechain resonances in these figures would require a minute font size. Therefore why we provided the assignments of the relevant regions of the same figures in enlarged versions displayed in Figs S5 and S8. A careful comparison of the zoomed regions with the overview figures revealed an error in spectral calibration, which we amended throughout."

I don’t see that the lettering of Trp sidechain assignments in Figures S3, S4, S7 would have to be any smaller than for the other annotated resonances. There is plenty of space and the annotation will make the data easier to understand for readers.

I would thus be happy to accept a suitably revised manuscript which takes into account these minor requests for improvement.

With best wishes
Stephan

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AR by Gottfried Otting on behalf of the Authors (20 Dec 2021) Author's response Author's tracked changes Manuscript

ED: Publish subject to corrections (21 Dec 2021) by Stephan Grzesiek

AR by Gottfried Otting on behalf of the Authors (22 Dec 2021) Author's response Manuscript

Short summary

This paper explores a method for determining the solution structure of a solvent-exposed polypeptide segment (the L3 loop), which is next to the active site of the penicillin-degrading enzyme IMP-1. Tagging three different sites on the protein with paramagnetic metal ions allowed positioning of the L3 loop with atomic resolution. It was found that the method was more robust when omitting data obtained with different metal ions if obtained with the same tag at the same tagging site.