Electric-field-stimulated protein mechanics

Abstract

The internal mechanics of proteins—the coordinated motions of amino acids and the pattern of forces constraining these motions—connects protein structure to function. Here we describe a new method combining the application of strong electric field pulses to protein crystals with time-resolved X-ray crystallography to observe conformational changes in spatial and temporal detail. Using a human PDZ domain (LNX2PDZ2) as a model system, we show that protein crystals tolerate electric field pulses strong enough to drive concerted motions on the sub-microsecond timescale. The induced motions are subtle, involve diverse physical mechanisms, and occur throughout the protein structure. The global pattern of electric-field-induced motions is consistent with both local and allosteric conformational changes naturally induced by ligand binding, including at conserved functional sites in the PDZ domain family. This work lays the foundation for comprehensive experimental study of the mechanical basis of protein function.

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Figure 1: EF-X principles and implementation.
Figure 2: An EF-X experiment in the LNX2PDZ2 domain.
Figure 3: The up–down internal difference analysis.
Figure 4: A gallery of electric field-induced structural effects.
Figure 5: The relationship between electric-field-induced conformational change and PDZ function.

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Acknowledgements

R.R. dedicates this paper to Alfred G. Gilman, whose contributions were profound and irreplaceable. We thank the staff at BioCARS, Stanford Synchrotron Radiation Lightsource (SSRL) and the UT Southwestern Medical Center Structural Biology Laboratory for technical support, and D. Borek, C. A. Brautigam, S. Leibler, A. Libchaber, K. Moffat, Z. Otwinowski and members of the Ranganathan laboratory for discussions. R.R. acknowledges support from National Institutes of Health (NIH) grant R01GM123456, the Robert A. Welch Foundation (I-1366), the Lyda Hill Endowment for Systems Biology, and the Green Center for Systems Biology. BioCARS is supported by NIH grant R24GM111072 and through a collaboration with P. Anfinrud (NIH/ National Institute of Diabetes and Digestive and Kidney Diseases). The SSRL is supported by the US Department of Energy (Contract No. DE-AC02-76SF00515) and by the NIH (P41GM103393).

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Authors

Contributions

D.R.H. and R.R. conceived the experimental approach. All authors contributed to the experimental design, D.R.H. and K.I.W. built the EF-X apparatus, and D.R.H., K.I.W., M.A.S. and R.R. performed experiments. D.R.H., V.S. and R.R. developed analysis methods and analysed the data. D.R.H. and R.R. wrote the manuscript with input from the other authors.

Corresponding author

Correspondence to Rama Ranganathan.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 The experimental setup of EF-X.

a, A plot relating the applied voltage across a 100-μm-thick crystal (left axis) and the size of transition dipole moments of conformational changes that can be excited by 1kBT (right axis) to the duration of the applied electric field. Feasible methods of generating strong electric field pulses are indicated as green and cyan shaded areas. Waveform and pulse generators can provide pulses down to the nanosecond timescale. Faster pulses can be generated using terahertz pulsed lasers with strong electric field components51 or by optical gating of semiconductors52; such systems are already present at third-generation synchrotron and X-ray free-electron laser facilities. The black bar indicates the approximate range covered by the current experiments. The calculation of temperature jumps caused by the electric field is described in Supplementary Information IA. b, Schematic cross-section of the counter electrode. The blue arrow indicates the path by which backpressure is applied to drive flow through the capillary (see Methods). c, Crystals are mounted on top of capillaries containing a metal electrode and soaked in crystallization solution. d, The capillary with crystal is mounted in a reusable goniometer base and protected from humidity fluctuations with a polyester sleeve (MiTeGen) containing 50% (v/v) crystallization solution. This assembly forms the bottom electrode. e, The counter and bottom electrodes are assembled at the beam line to allow rotation around the capillary axis. f, Once the sleeve is trimmed to just above the level of the crystal, the counter electrode is brought in using a translation stage (camera view of the approach) (Supplementary Video 2). g, Overview of the final set up with the direction of the X-ray and electric field pulses, reproduced from Fig. 1e.

Extended Data Figure 2 Tolerance of electric field pulses in several protein crystals.

a, Diffraction quality of a LNX2PDZ2 crystal (experiment 3-35, Supplementary Table 2), measured by the ratio of structure factor amplitude to noise (F/σ(F)) as a function of number of 250 ns, 6 kV electric field pulses and as a function of resolution bin (in colours, see legend). bd, Diffraction images for three other protein crystals before (left) and after (right) ~500 electric field pulses (precise value indicated). Crystal orientations are different between before and after frames. The data correspond to the following experiments in Supplementary Table 2: b, lysozyme, experiment 3-08; c, PDZ1 of PICK1, experiment 3-17; d, NaK2K, experiment 3-80. The data indicate that several protein crystals can tolerate the EF-X experiment.

Extended Data Figure 3 Internal consistency and temporal evolution of internal difference map signal.

ac, Analysis for the data presented in Fig. 3. a, Consistency of estimated signal per residue derived from two data collection passes on the same crystal (black: OFF; blue: 50 ns; green: 100 ns; red: 200 ns). Overall correlation coefficient 0.59. Signal is defined as the integrated absolute difference density above 2.5σOFF within 1.5 Å of the protein backbone, square-root transformed to stabilize variance. Per-time-point correlation coefficients are: −0.07 (OFF, P > 0.1), 0.23 (50 ns; P = 0.01); 0.35 (100 ns; P < 10−3) and 0.34 (200 ns; P < 10−3). b, Consistency of the obtained signal per residue between time points. Correlation coefficients are: 0.17 (OFF; P = 0.05), 0.55 (50 ns; P = 1 × 10−9) and 0.72 (100 ns; P < 10−20). The diagonal is shown for reference. Note that slight correlation in the OFF data set may indicate imperfect correction for anisotropic absorption. c, Signal integrated along the entire protein backbone in passes 1 and 2 (blue crosses and circles, respectively) and over the entire data set (squares). The red line indicates a naive expectation of a -fold increase in signal-to-noise ratio.

Extended Data Figure 4 Validation of signal in structure factors and difference maps.

ac, A negative correlation between and is consistent with oppositely directed motions in the up and down states. Analysis is performed over 20 resolution bins to allow for statistical testing. Shown are the correlation coefficients per bin between ΔFhkl and . In a linear response approximation and in the absence of measurement error, we expect . Reflections with |ΔFhkl| < σFhkl), or |ΔFhkl| > 10 were excluded from analysis, and likewise for . Results at 50 ns (a), 100 ns (b) and 200 ns (c). To assess significance, each bin was considered statistically homogeneous, with observations considered independent. Bins with significant negative deviation from 0 (after Fisher Z-transform) are indicated as filled circles (P < 10−3: black; P < 10−2: light blue). Error bars indicate standard errors based on the assumption of a normal distribution after Fisher Z-transform. df, Statistical significance of Fig. 3g. Comparison of integrated absolute difference density above 2.5σ, within 1.5 Å of backbone C, N and O atoms (‘signal’; see also Fig. 3a). d, Comparison of signal in the OFF state and at 50 ns. The grey-shaded area indicates the 0–95th percentile for random sampling from the OFF map at the same probe volume (because the conformation of the backbone changes from residue to residue, the effective probe volume varies along the protein backbone) and threshold. The blue-shaded area indicates the 0–95th percentile for random sampling using the conservative sampling protocol described in test 4 of the statistical validation. e, Same analysis at 100 ns. f, Same analysis at 200 ns. Note that we were unable to scale all diffraction images at once, and instead scaled the OFF data with each time point separately. We compare each ON data set to the OFF data as scaled with that time point. As a result, there are small differences between the OFF traces in df. gj, Deviations from a normal distribution for internal difference maps. Shown are voxel histograms for internal difference electron density (DED) maps without applied field (OFF) (g), and at 50 ns (h), 100 ns (i) and 200 ns (j) of applied electric field. Red lines indicate fits to a normal distribution based on calculated variance. Blue lines are histograms of voxel internal DED values (map grids of 0.3 Å). Note that by construction, for internal DED maps the positive and negative sides of the histogram are the same, apart from discretization effects. To assess statistical significance of deviations from normality, we sampled C2 asymmetric units (ASUs) at the Nyquist sampling frequency (here, 0.9 Å). For the OFF map, we find no significant deviations from normality (P > 0.1 for the Jarque–Bera test, the Anderson–Darling test, and the Lilliefors test; all using default settings in Matlab). At 200 ns, each test rejects a normal distribution with P < 0.01. At 50 and 100 ns, the results of statistical testing depend on how the internal DED map is subsampled: for a single C2 ASU, none of the tests rejects the null hypothesis, but when the same number of points is sampled from two neighbouring ASUs, the Jarque–Bera test rejects normality (P < 0.01 at 50 and 100 ns), suggesting limited deviation from normality. k, l, Reproducibility of a structural response to electric field. Correlation of data set 2 (see Supplementary Table 7) to the data set presented in the text. On the basis of ordinary differences ΔFhkl (k) and internal differences (l), reflections with |ΔFhkl| < σFhkl), , or |ΔFhkl| > 10 were excluded from analysis, and likewise for . The standard error of correlation coefficient estimates is ~0.07 in k and ~0.10 in l. Each bin is statistically homogeneous and observations are considered independent. Resolution bins with significant positive deviation from 0 (after Fisher Z-transform) are indicated as filled circles (P < 10−3: black; P < 10−2: light blue).

Extended Data Figure 5 Refinement, voltage-ON model at 200 ns.

a, Progress of refinement against extrapolated structure factors. Rounds marked by asterisks involved automated refinement with mild stereochemistry constraints to reduce deviations from optimal geometry due to manual refinement in Coot. Fluctuations in Rwork appear to be mostly due to the PHENIX bulk solvent scaling calculation used in R factor calculation. b, R factor for comparison of extrapolated structure factors, as a function of the degree of extrapolation, N, as derived from data set 2 (150 ns; see Supplementary Table 7), against calculated structure factors (Fc) derived from (1) the OFF model (black), (2) the excited state model (ESM) (red), and (3) an ‘upside-down’ ESM obtained by 180° rotation around the C2 two-fold rotation axis (blue), all derived from data set 1 (Extended Data Table 2). N relates to the fraction f of OFF signal subtracted as N = 1/(1 − f). No refinement against data set 2 was performed except for bulk solvent scaling. No test set was assigned. c, For comparison, the same analysis as in b, comparing the OFF model and 200 ns ESM model to the 100 ns data (from the same crystal). df, Relationship between Cα displacements in the up and down conformations at 200 ns. d, e, Projection of the down displacement on the direction of the up displacement (d), and the up displacement on the down displacement direction (all displacements are relative to the OFF model (e); models were superimposed using PyMOL, using C, Cα and N atoms of the protein backbone and including only residues 338–356, 362–380, 384–408 and 412–419; this excludes N- and C-terminal regions and mobile parts of the β2–β3, α1–β4 and α2–β6 loops). Shown are, for example Δ rdown · Δ rup/||Δ rup|| versus ||Δ rup||, as illustrated in the inset. For small displacements, a simple inverse dependence is expected. This is tested by robust linear regression for (projected) displacements smaller than 0.4 Å (red line fits to data in grey boxes; using default settings in Matlab). d, Slope = −0.80 ± 0.16, intercept = 0.081 ± 0.031 Å; correlation coefficient: −0.44. e, Slope = −0.41 ± 0.17, intercept = 0.012 ± 0.033 Å; correlation coefficient: −0.27. f, Average cosine between displacements of nearby Cα atoms as a function of distance along the primary structure.

Extended Data Figure 6 Additional views of conformational changes due to the electric field.

a, Reference model indicating regions examined in bf. bf, Maps and models as in Fig. 4, with motions indicated by arrows and residues coupled to ligand binding in PDZ domains shown (as in Supplementary Table 1). b, Top view of the α1 helix, waters omitted and the side chain of Q377 truncated for clarity. c, Transverse shift of the α2–β6 loop, and perturbed down state of S410, forming new hydrogen bonds to R413 and N391 (dashed blue lines). d, Upward motion of the β2–β3 loop and change in dynamic disorder of protein and solvent. e, Conformational changes at the top of the ligand-binding pocket, with motion of the terminal amine of the K344 towards the ligand carboxylate group in the down state. f, Coupled rotameric changes of L402 (α2 helix), L395 and D394.

Extended Data Figure 7 Biasing pre-existing conformational heterogeneity in the LNX2PDZ2 ground state structure by the external electric field: additional examples.

a, b, A high-resolution (1.1 Å) room-temperature structure of the voltage-OFF ground state of LNX2PDZ2 (Extended Data Table 3), shows partial occupancy of N415 (a) and D368 (b) in two rotameric states (left). This pre-existing conformational equilibrium is biased in the presence of the electric field (6 kV, 200 ns delay), such that the up and down models each adopt one of the two ground state configurations (middle and right). This supports the result shown in Fig. 4g.

Extended Data Table 1 Estimates of dipole moments associated with conformational changes
Extended Data Table 2 Data collection and refinement statistics for LNX2PDZ2 for EF-X experiment
Extended Data Table 3 Data collection and refinement statistics for LNX2PDZ2 by room-temperature crystallography

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Supplementary Tables 1-7, full legends for PyMol Session files 1-4 and Supplementary References – see contents page for full details. (PDF 4250 kb)

Supplementary Data

This file contains PyMol Session S1, internal electron density difference map shown on the OFF structure – see Supplementary Information document for full description. (ZIP 24995 kb)

Supplementary Data

This file contains PyMol Session S2, superimposed up, down, and OFF models and 2Fo-Fc electron density – see Supplementary Information document for full description. (ZIP 17704 kb)

Supplementary Data

This file contains PyMol Session S3, superimposed up, down, and OFF models and 2Fo-Fc electron density for a composite omit map calculated with iterative refinement – see Supplementary Information document for full description. (ZIP 16572 kb)

Supplementary Data

This file contains PyMol Session S4, 2Fo-Fc electron density obtained for refinement against extrapolated structure factors in the reduced symmetry (P1) space group – see Supplementary Information document for full description. (ZIP 33680 kb)

Destructive breakdown after dielectric seal failure

An experiment in which misalignment of electrodes led to dissociation of the protein crystal (lysozyme) from the bottom electrode, providing a conductive path around the crystal and leading to destructive arcing (dielectric breakdown). The video was recorded from a monitor with a piece of transparent adhesive tape with red dot indicating beam center, used in sample alignment. (MOV 3452 kb)

Establishing liquid junction between top counter electrode and the crystal

The approach of the top electrode by manual control of a translation stage. The electrode is brought in sufficiently close to establish a liquid junction between the top electrode and the crystal. (MOV 27777 kb)

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Hekstra, D., White, K., Socolich, M. et al. Electric-field-stimulated protein mechanics. Nature 540, 400–405 (2016). https://doi.org/10.1038/nature20571

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