Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Temperature-jump solution X-ray scattering reveals distinct motions in a dynamic enzyme

Abstract

Correlated motions of proteins are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved X-ray methods hold promise for addressing this challenge, but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature jumps, through thermal excitation of the solvent, have been utilized to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform temperature-jump small- and wide-angle X-ray scattering measurements on a dynamic enzyme, cyclophilin A, demonstrating that these experiments are able to capture functional intramolecular protein dynamics on the microsecond timescale. We show that cyclophilin A displays rich dynamics following a temperature jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes. Two relaxation processes are resolved: a fast process is related to surface loop motions, and a slower process is related to motions in the core of the protein that are critical for catalytic turnover.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Overview of temperature-jump SAXS/WAXS experiments.
Fig. 2: Temperature-jump data allow kinetic modelling of conformational dynamics.
Fig. 3: Time-resolved Guinier analysis.
Fig. 4: Eyring analysis of transition-state thermodynamics.
Fig. 5: Kinetic analysis of CypA variants.

Data availability

Scattering data are deposited at NIH Figshare (https://doi.org/10.35092/yhjc.9177143). Additional information and files are available from the corresponding author upon reasonable request.

Code availability

All Python scripts used for analysis of integrated X-ray scattering curves are publicly available from GitHub (https://github.com/fraser-lab/solution_scattering). A code release checkpoint containing the exact scripts used in this work is available via Zenodo (https://doi.org/10.5281/zenodo.3355707).

References

  1. Henzler-Wildman, K. & Kern, D. Dynamic personalities of proteins. Nature 450, 964–972 (2007).

    CAS  PubMed  Google Scholar 

  2. Van den Bedem, H. & Fraser, J. S. Integrative, dynamic structural biology at atomic resolution—it’s about time. Nat. Methods 12, 307–318 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Bottaro, S. & Lindorff-Larsen, K. Biophysical experiments and biomolecular simulations: a perfect match? Science 361, 355–360 (2018).

    CAS  PubMed  Google Scholar 

  4. Schmidt, M. Time-resolved macromolecular crystallography at modern X-ray sources. Methods Mol. Biol. 1607, 273–294 (2017).

    CAS  PubMed  Google Scholar 

  5. Neutze, R. & Moffat, K. Time-resolved structural studies at synchrotrons and X-ray free electron lasers: opportunities and challenges. Curr. Opin. Struct. Biol. 22, 651–659 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Schotte, F. et al. Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography. Proc. Natl Acad. Sci. USA 109, 19256–19261 (2012).

    CAS  PubMed  Google Scholar 

  7. Cho, H. S. et al. Picosecond photobiology: watching a signaling protein function in real time via time-resolved small- and wide-angle X-ray scattering. J. Am. Chem. Soc. 138, 8815–8823 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Schlichting, I. & Miao, J. Emerging opportunities in structural biology with X-ray free-electron lasers. Curr. Opin. Struct. Biol. 22, 613–626 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Arnlund, D. et al. Visualizing a protein quake with time-resolved X-ray scattering at a free-electron laser. Nat. Methods 11, 923–926 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Berntsson, O. et al. Sequential conformational transitions and α-helical supercoiling regulate a sensor histidine kinase. Nat. Commun. 8, 284 (2017).

    PubMed  PubMed Central  Google Scholar 

  11. Takala, H. et al. Signal amplification and transduction in phytochrome photosensors. Nature 509, 245–248 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Brinkmann, L. U. L. & Hub, J. S. Ultrafast anisotropic protein quake propagation after CO photodissociation in myoglobin. Proc. Natl Acad. Sci. USA 113, 10565–10570 (2016).

    CAS  PubMed  Google Scholar 

  13. Barends, T. R. M. et al. Direct observation of ultrafast collective motions in CO myoglobin upon ligand dissociation. Science 350, 445–450 (2015).

    CAS  PubMed  Google Scholar 

  14. Coquelle, N. et al. Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography. Nat. Chem. 10, 31–37 (2018).

    CAS  PubMed  Google Scholar 

  15. Pande, K. et al. Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein. Science 352, 725–729 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Kern, J. et al. Structures of the intermediates of Kok’s photosynthetic water oxidation clock. Nature 563, 421–425 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Nogly, P. et al. Retinal isomerization in bacteriorhodopsin captured by a femtosecond X-ray laser. Science 361, eaat0094 (2018).

    PubMed  Google Scholar 

  18. Malmerberg, E. et al. Time-resolved WAXS reveals accelerated conformational changes in iodoretinal-substituted proteorhodopsin. Biophys. J. 101, 1345–1353 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Hekstra, D. R. et al. Electric-field-stimulated protein mechanics. Nature 540, 400–405 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Schlichting, I. et al. Time-resolved X-ray crystallographic study of the conformational change in Ha-Ras p21 protein on GTP hydrolysis. Nature 345, 309–315 (1990).

    CAS  PubMed  Google Scholar 

  21. Stoddard, B. L., Cohen, B. E., Brubaker, M., Mesecar, A. D. & Koshland, D. E. Jr Millisecond Laue structures of an enzyme-product complex using photocaged substrate analogs. Nat. Struct. Biol. 5, 891–897 (1998).

    CAS  PubMed  Google Scholar 

  22. Josts, I. et al. Photocage-initiated time-resolved solution X-ray scattering investigation of protein dimerization. IUCrJ 5, 667–672 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Rimmerman, D. et al. Revealing fast structural dynamics in pH-responsive peptides with time-resolved X-ray scattering. J. Phys. Chem. B 123, 2016–2021 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Olmos, J. L. Jr et al. Enzyme intermediates captured ‘on the fly’ by mix-and-inject serial crystallography. BMC Biol. 16, 59 (2018).

    PubMed  PubMed Central  Google Scholar 

  25. Rimmerman, D. et al. Direct observation of insulin association dynamics with time-resolved X-ray scattering. J. Phys. Chem. Lett. 8, 4413–4418 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Rimmerman, D. et al. Insulin hexamer dissociation dynamics revealed by photoinduced T-jumps and time-resolved X-ray solution scattering. Photochem. Photobiol. Sci. 17, 874–882 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Cho, H. S. et al. Dynamics of quaternary structure transitions in R-state carbonmonoxyhemoglobin are unveiled in time-resolved X-ray scattering patterns following a temperature jump. J. Phys. Chem. B 122, 11488–11496 (2018).

    CAS  PubMed  Google Scholar 

  28. Frauenfelder, H., Fenimore, P. W. & Young, R. D. Protein dynamics and function: insights from the energy landscape and solvent slaving. IUBMB Life 59, 506–512 (2007).

    CAS  PubMed  Google Scholar 

  29. Fenimore, P. W., Frauenfelder, H., McMahon, B. H. & Parak, F. G. Slaving: solvent fluctuations dominate protein dynamics and functions. Proc. Natl Acad. Sci. USA 99, 16047–16051 (2002).

    CAS  PubMed  Google Scholar 

  30. Wang, J. & El-Sayed, M. A. Temperature jump-induced secondary structural change of the membrane protein bacteriorhodopsin in the premelting temperature region: a nanosecond time-resolved Fourier transform infrared study. Biophys. J. 76, 2777–2783 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Wang, T., Lau, W. L., DeGrado, W. F. & Gai, F. T-jump infrared study of the folding mechanism of coiled-coil GCN4-p1. Biophys. J. 89, 4180–4187 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Akasaka, K., Naito, A. & Nakatani, H. Temperature-jump NMR study of protein folding: ribonuclease A at low pH. J. Biomol. NMR 1, 65–70 (1991).

    CAS  PubMed  Google Scholar 

  33. Gillespie, B. et al. NMR and temperature-jump measurements of de novo designed proteins demonstrate rapid folding in the absence of explicit selection for kinetics. J. Mol. Biol. 330, 813–819 (2003).

    CAS  PubMed  Google Scholar 

  34. Yamasaki, K. et al. Real-time NMR monitoring of protein-folding kinetics by a recycle flow system for temperature jump. Anal. Chem. 85, 9439–9443 (2013).

    CAS  PubMed  Google Scholar 

  35. Meadows, C. W., Balakrishnan, G., Kier, B. L., Spiro, T. G. & Klinman, J. P. Temperature-jump fluorescence provides evidence for fully reversible microsecond dynamics in a thermophilic alcohol dehydrogenase. J. Am. Chem. Soc. 137, 10060–10063 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Vaughn, M. B., Zhang, J., Spiro, T. G., Dyer, R. B. & Klinman, J. P. Activity-related microsecond dynamics revealed by temperature-jump Förster resonance energy transfer measurements on thermophilic alcohol dehydrogenase. J. Am. Chem. Soc. 140, 900–903 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Hori, T. et al. The initial step of the thermal unfolding of 3-isopropylmalate dehydrogenase detected by the temperature-jump Laue method. Protein Eng. 13, 527–533 (2000).

    CAS  PubMed  Google Scholar 

  38. Eisenmesser, E. Z. et al. Intrinsic dynamics of an enzyme underlies catalysis. Nature 438, 117–121 (2005).

    CAS  PubMed  Google Scholar 

  39. Caines, M. E. C. et al. Diverse HIV viruses are targeted by a conformationally dynamic antiviral. Nat. Struct. Mol. Biol. 19, 411–416 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Price, A. J. et al. Active site remodeling switches HIV specificity of antiretroviral TRIMCyp. Nat. Struct. Mol. Biol. 16, 1036–1042 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Virgen, C. A., Kratovac, Z., Bieniasz, P. D. & Hatziioannou, T. Independent genesis of chimeric TRIM5-cyclophilin proteins in two primate species. Proc. Natl Acad. Sci. USA 105, 3563–3568 (2008).

    CAS  PubMed  Google Scholar 

  42. Wilson, S. J. et al. Independent evolution of an antiviral TRIMCyp in rhesus macaques. Proc. Natl Acad. Sci. USA 105, 3557–3562 (2008).

    CAS  PubMed  Google Scholar 

  43. Keedy, D. A. et al. Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography.eLife 4, e07574 (2015).

    PubMed  PubMed Central  Google Scholar 

  44. Fraser, J. S. et al. Hidden alternative structures of proline isomerase essential for catalysis. Nature 462, 669–673 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Otten, R. et al. Rescue of conformational dynamics in enzyme catalysis by directed evolution. Nat. Commun. 9, 1314 (2018).

    PubMed  PubMed Central  Google Scholar 

  46. Clark, G. N. I., Hura, G. L., Teixeira, J., Soper, A. K. & Head-Gordon, T. Small-angle scattering and the structure of ambient liquid water. Proc. Natl Acad. Sci. USA 107, 14003–14007 (2010).

    CAS  PubMed  Google Scholar 

  47. Kjær, K. S. et al. Introducing a standard method for experimental determination of the solvent response in laser pump, X-ray probe time-resolved wide-angle X-ray scattering experiments on systems in solution. Phys. Chem. Chem. Phys. 15, 15003–15016 (2013).

    PubMed  Google Scholar 

  48. Cammarata, M. et al. Impulsive solvent heating probed by picosecond X-ray diffraction. J. Chem. Phys. 124, 124504 (2006).

    CAS  PubMed  Google Scholar 

  49. Gruebele, M., Sabelko, J., Ballew, R. & Ervin, J. Laser temperature jump induced protein refolding. Acc. Chem. Res. 31, 699–707 (1998).

    CAS  Google Scholar 

  50. Levantino, M. et al. Ultrafast myoglobin structural dynamics observed with an X-ray free-electron laser. Nat. Commun. 6, 6772 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. DiCiccio, T. J. & Efron, B. Bootstrap confidence intervals. Stat. Sci. 11, 189–212 (1996).

    Google Scholar 

  52. Smith, C. A. et al. Population shuffling of protein conformations. Angew. Chem. Int. Ed. Engl. 54, 207–210 (2015).

    CAS  PubMed  Google Scholar 

  53. Wapeesittipan, P., Mey, A., Walkinshaw, M. & Michel, J. Allosteric effects in catalytic impaired variants of the enzyme cyclophilin A may be explained by changes in nano-microsecond time scale motions. Commun. Chem. 2, 41 (2019).

    Google Scholar 

  54. Chi, C. N. et al. A structural ensemble for the enzyme cyclophilin reveals an orchestrated mode of action at atomic resolution. Angew. Chem. Int. Ed. Engl. 54, 11657–11661 (2015).

    CAS  PubMed  Google Scholar 

  55. Kim, J. G. et al. Cooperative protein structural dynamics of homodimeric hemoglobin linked to water cluster at subunit interface revealed by time-resolved X-ray solution scattering. Struct. Dynam. 3, 023610 (2016).

    Google Scholar 

  56. Colombo, M. F., Rau, D. C. & Parsegian, V. A. Protein solvation in allosteric regulation: a water effect on hemoglobin. Science 256, 655–659 (1992).

    CAS  Google Scholar 

  57. Salvay, A. G., Grigera, J. R. & Colombo, M. F. The role of hydration on the mechanism of allosteric regulation: in situ measurements of the oxygen-linked kinetics of water binding to hemoglobin. Biophys. J. 84, 564–570 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Royer, W. E. Jr, Pardanani, A., Gibson, Q. H., Peterson, E. S. & Friedman, J. M. Ordered water molecules as key allosteric mediators in a cooperative dimeric hemoglobin. Proc. Natl Acad. Sci. USA 93, 14526–14531 (1996).

    CAS  PubMed  Google Scholar 

  59. Fenwick, R. B., Oyen, D., Dyson, H. J. & Wright, P. E. Slow dynamics of tryptophan–water networks in proteins. J. Am. Chem. Soc. 140, 675–682 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Grossman, M. et al. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site. Nat. Struct. Mol. Biol. 18, 1102–1108 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Decaneto, E. et al. Solvent water interactions within the active site of the membrane type I matrix metalloproteinase. Phys. Chem. Chem. Phys. 19, 30316–30331 (2017).

    CAS  PubMed  Google Scholar 

  62. Leidner, F., Kurt Yilmaz, N., Paulsen, J., Muller, Y. A. & Schiffer, C. A. Hydration structure and dynamics of inhibitor-bound HIV-1 protease. J. Chem. Theory Comput. 14, 2784–2796 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Guha, S. et al. Slow solvation dynamics at the active site of an enzyme: implications for catalysis. Biochemistry 44, 8940–8947 (2005).

    CAS  PubMed  Google Scholar 

  64. Dahanayake, J. N. & Mitchell-Koch, K. R. Entropy connects water structure and dynamics in protein hydration layer. Phys. Chem. Chem. Phys. 20, 14765–14777 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Wand, A. J. & Sharp, K. A. Measuring entropy in molecular recognition by proteins. Annu. Rev. Biophys. 47, 41–61 (2018).

    CAS  PubMed  Google Scholar 

  66. Caro, J. A. et al. Entropy in molecular recognition by proteins. Proc. Natl Acad. Sci. USA 114, 6563–6568 (2017).

    CAS  PubMed  Google Scholar 

  67. Gavrilov, Y., Leuchter, J. D. & Levy, Y. On the coupling between the dynamics of protein and water. Phys. Chem. Chem. Phys. 19, 8243–8257 (2017).

    CAS  PubMed  Google Scholar 

  68. Conti Nibali, V., D’Angelo, G., Paciaroni, A., Tobias, D. J. & Tarek, M. On the coupling between the collective dynamics of proteins and their hydration water. J. Phys. Chem. Lett. 5, 1181–1186 (2014).

    CAS  PubMed  Google Scholar 

  69. Hub, J. S. Interpreting solution X-ray scattering data using molecular simulations. Curr. Opin. Struct. Biol. 49, 18–26 (2018).

    CAS  PubMed  Google Scholar 

  70. Svergun, D. I. et al. Protein hydration in solution: experimental observation by X-ray and neutron scattering. Proc. Natl Acad. Sci. USA 95, 2267–2272 (1998).

    CAS  PubMed  Google Scholar 

  71. Virtanen, J. J., Makowski, L., Sosnick, T. R. & Freed, K. F. Modeling the hydration layer around proteins: applications to small- and wide-angle X-ray scattering. Biophys. J. 101, 2061–2069 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Henriques, J., Arleth, L., Lindorff-Larsen, K. & Skepö, M. On the calculation of SAXS profiles of folded and intrinsically disordered proteins from computer simulations. J. Mol. Biol. 430, 2521–2539 (2018).

    CAS  PubMed  Google Scholar 

  73. Dellus-Gur, E. et al. Negative epistasis and evolvability in TEM-1 β-lactamase—the thin line between an enzyme’s conformational freedom and disorder. J. Mol. Biol. 427, 2396–2409 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Biel, J. T., Thompson, M. C., Cunningham, C. N., Corn, J. E. & Fraser, J. S. Flexibility and design: conformational heterogeneity along the evolutionary trajectory of a redesigned ubiquitin. Structure 25, 739–749.e3 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Fischer, M., Coleman, R. G., Fraser, J. S. & Shoichet, B. K. Incorporation of protein flexibility and conformational energy penalties in docking screens to improve ligand discovery. Nat. Chem. 6, 575–583 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Fischer, M., Shoichet, B. K. & Fraser, J. S. One crystal, two temperatures: cryocooling penalties alter ligand binding to transient protein sites. ChemBioChem 16, 1560–1564 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Keedy, D. A. et al. An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering. eLife 7, e36307 (2018).

    PubMed  PubMed Central  Google Scholar 

  78. Fraser, J. S. et al. Accessing protein conformational ensembles using room-temperature X-ray crystallography. Proc. Natl Acad. Sci. USA 108, 16247–16252 (2011).

    CAS  PubMed  Google Scholar 

  79. Van den Bedem, H., Bhabha, G., Yang, K., Wright, P. E. & Fraser, J. S. Automated identification of functional dynamic contact networks from X-ray crystallography. Nat. Methods 10, 896–902 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank R. Ranganathan, J. Holton, G. Hura and D. Elnatan for helpful discussions, and the staff at the BioCARS beamline at the Advanced Photon Source (I. Kosheleva, R. Henning, A. DiChiara and V. Srajer) for assistance. This work was supported by the NSF (STC-1231306), the NIH (GM123159 and GM124149), a Packard Fellowship from the David and Lucile Packard Foundation, the UC Office of the President Laboratory Fees Research Program LFR-17-476732 (to J.S.F.), the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (to P.A.) and a Ruth L. Kirschstein National Research Service Award (F32 HL129989 to M.C.T.). Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract number DE-AC02-06CH11357. Use of the BioCARS Sector 14 was also supported by the NIH National Institute of General Medical Sciences (grant R24GM111072). The time-resolved setup at Sector 14 was funded in part through a collaboration with P.A. (NIH/NIDDK).

Author information

Authors and Affiliations

Authors

Contributions

M.C.T., P.A. and J.S.F. conceived and designed the experiments. M.C.T., B.A.B., A.M.W., H.S.C., F.S., D.M.C.S. and P.A. performed the experiments. M.C.T., B.A.B. and A.M.W. analysed the data. M.C.T., B.A.B., A.M.W., H.S.C., F.S. and P.A. contributed materials/analysis tools. M.C.T. and J.S.F. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Philip Anfinrud or James S. Fraser.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8, Supplementary Table 1 and Supplementary Data, Methods and References.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Thompson, M.C., Barad, B.A., Wolff, A.M. et al. Temperature-jump solution X-ray scattering reveals distinct motions in a dynamic enzyme. Nat. Chem. 11, 1058–1066 (2019). https://doi.org/10.1038/s41557-019-0329-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-019-0329-3

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing