supporting information - pnas · supporting information ... analytical sec was performed on an akta...

Supporting InformationGupta et al. 10.1073/pnas.1512240112SI MethodsProtein Purification.OCP was purified from an OCP overexpressingmutant strain of Synechocystis PCC 6803 (a gift of Diana Kirilovsky,Commissariat à l’Energie Atomique, Institut de Biologie et Tech-nologies de Saclay, Gif-sur-Yvette, France) using a proceduresimilar to that described previously (7). In the Synechocystis PCC6803 strain used for protein expression, the CrtR gene (responsiblefor carotenoid hydroxylation) is interrupted by an antibiotic re-sistance cassette, and its OCP is known to bind predominantlyECN (34). Modifications to the previously described OCP purifi-cation procedure included the following: (i) lysis by bead-beating(9 × 20-s cycles at 0 °C), (ii) intermediate purification by ion-exchange chromatography (IEC) on a HiPrep Q-Sepharose HP 16/10column (GE Healthcare), and (iii) a final gel filtration purificationstep after IEC using a Superdex 75 16/60 SEC column and 50 mMTris·HCl (pH 7.5), 200 mM NaCl running buffer. Sample purity wasqualified by SDS/PAGE and the UV-visible absorbance spectrum ofthe purified holoprotein. OCP was buffer exchanged by gel filtrationbefore XF-MS and HDX experiments using a Superdex 75 10/300GL column (GE Healthcare) preequilibrated with 20 mM potassiumphosphate, 100 mM NaCl (pH 7.4).

Analytical SEC. Analytical SEC was performed on an AKTA PureM2 chromatography system (GE Healthcare) with triple wave-length absorbance detection. A set of globular standards (cat. no.151-1901; Bio-Rad) was used to calibrate the column for MWestimations by determining the best linear fit to a plot of partitioncoefficient versus log(MW) for three globular standards in thelinear fractionation range of the Superdex 200 10/300 GL column.Identical running conditions (100-μL injection volume, 0.75 mL/minflow rate, 4 °C run temperature) were used for both OCP samplesand standards in all buffers, and a separate standard calibrationcurve was calculated in each buffer. For SEC runs of OCPR, OCPwas preilluminated for 5 min on ice with a blue LED lamp (∼470 nmλmax, three LEDs per lamp, Luxeon Rebel LXML-PB01-0030LED; Lumileds).

Circular Dichroism.Measurement of far-UV CD spectra. OCP samples were exchanged into20 mM potassium phosphate, 100 mMNaF (pH 7.4) by gel filtrationon a Superdex 75 10/300 GL column before CD measurements.Sample concentrations ranged from 0.10 to 0.17 mg/mL for fourindependent replicates, and concentrations were determined usingthe maximum extinction coefficient of ECN (118,000 M-1·cm−1)(35) and the known 1:1 binding stoichiometry of ECN:OCP. OCPCD spectra in the far-UV range were collected in a 1-mm path-length quartz cuvette using a Chirascan-plus spectropolarimeter(Applied Photophysics). Individual scans were collected in the 260-to 185-nm range with 0.5-nm steps, 0.5 s per point, and 1-nmspectral bandwidth. Smoothed spectra were obtained by averagingeight scans, subtracting a smoothed background spectrum, and theapplication of a Savitsky–Golay filter to the averaged data. Units ofellipticity (in millidegrees) were converted to mean-residue-ellip-ticity as described in ref. 36. The OCPR CD spectrum was collectedafter illumination (5 min with a blue LED at 0 °C) of the samesample/cuvette used for the OCPO spectrum acquisition. UV-visi-ble absorption spectra collected before and after collection of theOCPR CD dataset confirmed that the sample remained predomi-nantly in the OCPR state during the time necessary to make theCD measurement (Fig. S2E). After OCPR CD measurements, thesample was allowed to thermally revert to OCPO at 24 °C. UV-visible absorption (Fig. S2E) and far-UV CD spectra collected

after thermal reversion confirmed that the light-induced changesobserved by CD were largely reversible.Secondary structure estimation. Estimations of protein secondarystructure were performed using CDSSTR, CONTINLL, andSELCON3 packages in the CDPro program suite (17) using fivedifferent reference sets of soluble protein CD spectra. Analyseswere performed on averaged and smoothed CD spectra (OCPR

and OCPO) from four independent experiments.

SAXS Data Collection and Analysis. SAXS measurements wereconducted on beamline 4-2 at the Stanford Synchrotron Radia-tion Lightsource. The focused 11-keV X-ray beam irradiated athin-wall quartz capillary cell, placed at 1.7 m upstream of theRayonix MX 225HE detector. One hundred microliters of OCP(1.3 mg/mL) were injected onto a high resolution Sepharose 200column (GE Healthcare) with a flow rate of 50 μL/min in 20 mMNa3PO4 (pH 7.4), 150 mM NaCl, 0.02% NaN3, 1 mM EDTA.The flow from the column passed through a UV detector cell,followed by 15 cm of clear tubing leading to the inlet of thequartz capillary cell. As the flow passed the quartz capillary, 1-sexposures were collected every 5 s throughout the run, with acirculating water bath maintaining the capillary cell temperatureat 8 °C. The detector pixel numbers were converted to the mo-mentum transfer q = 4p*sinq/l, where 2*q is the scattering angleand l the X-ray wavelength of 1.127 A°, using a silver behenatepowder standard placed at the capillary position (37). A back-ground scattering curve was obtained from the first 100 expo-sures (before the void volume), which was subtracted from allsubsequent exposures during the elution profile. Rg and I(0) foreach frame were batch-analyzed using autoRg (38), and frameswith stable Rg values were merged in PRIMUS (39) for the finalscattering curve.The inactive OCP dataset was collected by wrapping all clear

lines from the column in aluminum foil in the absence of anybackground light in the hutch. An additional dataset was collectedbypassing the UV detector to ensure that the UV cell was notcausing any activation of OCP (Fig. S3D). Activated OCP wasmeasured by placing one blue LED to illuminate the clear tubingleading to the capillary and a second blue LED to illuminate thecapillary just before the beam path. The duration of illuminationwith this setup was ∼20–25 seconds, and there was only 1–2 s be-tween illumination and X-ray exposure. The half-life of reversionof OCPR to OCPO is ∼30 s at 22 °C and much slower at lowertemperatures (7); therefore, no significant reversion should occurbefore X-ray exposure. The LEDs were placed either 20 cm away(weak illumination), 7 cm away (medium illumination), or 2 cmaway (strong illumination) from the lines and capillary. The LEDswere on for collection of the background scattering signal andturned on 5 min before elution of OCP from the column to min-imize the heating of the lines that can occur over longer illumi-nation times.The particle distance distribution function [P(r)] plot was

calculated from the merged scattering data using GNOM (40).The transformed data were subsequently used for generatingensembles of ab initio shape reconstructions usingDAMMIN v5.3(21) or GASBOR22i (20). The 12 calculated bead models werevery consistent with the scattering data as reflected by low χvalues (DAMMIN, 1.14 ± 0.01, 1.25 ± 0.02; GASBOR, 1.15 ±0.02, 1.31 ± 0.03, for OCPO and OCPR, respectively). The beadmodels were aligned using SUPCOMB13 with enantiomersconsidered (41), and spatially filtered using DAMFILT (42). Thenormalized spatial discrepancy (NSD) value for each ensemble

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was below 2, indicating good consistency between the individualbead models (DAMMIN, 0.64 ± 0.02, 0.55 ± 0.01; GASBOR,1.23 ± 0.03, 1.38 ± 0.07, for OCPO and OCPR, respectively).Domains of OCP were manually docked into the reconstructedshape volumes using Chimera (43).Coral (ATSAS package) was used for rigid body modeling

of the SAXS data for OCPR. The structure of the isolated N-terminal domain (9) (residues 18–165) and C-terminal domains(residues 185–306 from PDB ID code 3MG1) were treated asrigid bodies. The N termini (2–17), C terminus with the His tag(307–322), and linker region (166–184) were treated as flexibleportions. Ten calculations were performed with the default Coralsettings to sample various domain and linker orientations.

X-Ray Radiolysis and Mass Spectrometry. OCPECN (expressed inEscherichia coli) was exchanged into 20 mM potassium phos-phate (pH 7.4), 100 mMNaCl by SEC on a Superdex-75 10/300 GLcolumn before XF-MS experiments. Before X-ray irradiationof OCPR, the sample syringe was cooled with an ice pack and il-luminated with a blue LED array (470 nm Luxeon Rebel; PhilipsLumileds). Protein samples were irradiated in the microsecond timerange at beamline 5.3.1 at the Advanced Light Source as reportedpreviously (30). All samples, including the control (no X-ray irradi-ation), were subjected to cys-alkylation and de-salting before over-night trypsin and endoproteinase GluC digestions at pH 8 and37 °C. Proteolyzed samples were analyzed in an Agilent 6550iFunnel Q-TOF mass spectrometer (Agilent Technologies) coupledto an Agilent 1290 LC system (Agilent) using Sigma-Aldrich As-centis Peptides ES-C18 reverse phase column (2.1 mm × 100 mm,2.7-μm particle size; Sigma-Aldrich). The concentrations of thecomponents in the gradient are measured as vol/vol. Approximately10 pmol of samples were loaded onto the column via an InfinityAutosampler (Agilent) with buffer A (2% acetonitrile, 0.1% formicacid) flowing at 0.400 mL/min. The peptides were separated andeluted into the mass spectrometer via a gradient with initial condi-tion of 5% buffer B (98% acetonitrile, 0.1% formic acid) increasingto 70% B over 15 min. Subsequently, B was increased to 90% over1 min and held for 3 min at a flow rate of 0.6 mL/min, followedby a ramp back down to 5% over 1 min, where it was held forminutes to reequilibrate the column to the original condition.Peptides were introduced to the mass spectrometer from the LCusing a Jet Stream source (Agilent) operating in positive-ion mode(3,500 V). The data were acquired with MassHunter B.05.00 op-erating in Auto MS/MS mode whereby the three most intense ions(charge states 2–5) within 300 m/z to 1,400 m/z mass range above athreshold of 1,000 counts were selected for MS/MS analysis. MS/MS spectra were collected with the quadrupole set to “Narrow”resolution and collision energy to optimize fragmentation. MS/MSspectra were scanned from m/z 100–1,700 and were collected until40,000 total counts were collected or for a maximum accumulationtime of 333 ms. Parent ions were excluded for 0.1 min after MS/MSacquisition. MS/MS data of native and modified peptide fragmentswere interpreted by Mascot MS/MS Ions Search, as well as verifiedmanually. The abundance of native and modified peptides at anyirradiation time point area were measured (peak area) from theirrespective extracted ion chromatogram using Agilent MassHunter v 2.0.The peak area from the extracted ion chromatograms of a

specific peptide fragment with a particular mass-to-charge ratio

and associated +16-, +32-, or +48-Da side-chain modificationswas used to quantify the amount of modification at a given irra-diation time. Increasing irradiation progressively reduces the frac-tion of unmodified products and provides a site-specific dose–response plot (as in Fig. S5B). The hydroxyl radical reactivity rate(k), which depends on both intrinsic reactivity and solvent accessi-bility, was obtained by fitting the dose–response to a single expo-nential decay (based on a pseudo-first order reaction scheme usingOrigin 7.5 (OriginLabs). The ratio (R) of the measured reactivityof the side chains residues between OCPO and OCPR (R =kOCPO/kOCPR) gave information on solvent accessibility changesindependent of the intrinsic reactivity (Table S5).

Hydrogen–Deuterium Exchange and Mass Spectrometry. Ten mi-croliters of the OCP stock (0.5 mg/mL in PBS: 10 mMNaPO4, pH7.0, 150 mM NaCl, 10 mM DTT) was exposed to the blue LED(described in Analytical SEC) at 25 °C at a distance of 7 cm for 30 s,at which point 90 μL of buffered D2O (PBS in 95% D2O) wasadded. The sample remained illuminated through the course ofdeuterium exchange, after which the sample was transferred to atube on ice containing 100 μL of 0.4% formic acid with 0.15 mg/mLpepsin for a final pH of 2.5. The samples were digested on ice for5 min, flash frozen in liquid nitrogen, and stored at −80 °C untilLC-MS analysis. Sample heating from the LED at this distance wasless than 1 °C over a 30-min time course. Undeuterated sampleswere prepared identically substituting D2O for optima LC-MSgrade water. A fully deuterated sample was prepared by denaturinga solution of OCP at 0.5 mg/mL in 3 M GndHCl, heating at 85 °Cfor 30 min, diluting into D2O just as for the other samples, in-cubating for 1 h at 60 °C, then quenching just as for the othersamples. A “zero” time point to correct for “IN exchange” wasprepared by adding 10 μL of the OCP stock to prequenched D2Oand digesting just as for the other samples. An internal tetrapep-tide (PPPI) was included in all samples to ensure that both OCPO

and OCPR were exposed to the same deuterium exchange condi-tions (last plot in Fig. S3) (44).Deuterated samples were thawed and rapidly analyzed by LC-

MS on aWaters SynaptQ-TOFmass spectrometer. Peptides werecaptured and desalted on a trap column for 3 min (Vanguard BEH,1.7 μ 1 × 17 mm; Waters) and resolved on a 1 × 50-mm 2.1 μ C18Hypersil gold Thermo using a linear gradient of 5–40% B in 8 min(A, 0.05% TFA, 5% acetonitrile; B, 0.05% TFA 80% acetonitrile).The injection loop, columns, and lines were kept under melting iceto minimize back-exchange. For peptide identification, data-dependent MS/MS scans of the peptic digests were acquired onan Orbitrap LTQ (Thermo). MS/MS data along with exact massinformation were used to identify peptic fragments with the aidof protein prospector (prospector.ucsf.edu). Deuterium levelswere analyzed using HX-Express v2 (45), and percent exchange foreach time point (Dx) was calculated relative to the fully deuterated(DT) and zero (D0) standards [%D = (Dx − D0)/(DT − D0)].

Dynamic and Static Light Scattering. All experiments were per-formed on a Dynapro (Wyatt Technologies) with 30 acquisitionsof 10 s each at 4 °C, either in the absence of light (OCPO), or after1 min of illumination on ice (OCPR). The cuvette and tubes wereleft on ice before measurements to maintain the sample at lowtemperatures to minimize reversion back to the dark state, whichoccurs only with a half-life of >45 min at this temperature (7).


http://prospector.ucsf.edu/


Fig. S1. Structure of the OCP. (A) Crystal structure of Synechocystis OCP (PDB ID code 3MG1) consisting of two domains, NTD and CTD as described in the maintext introduction, which form major and minor interfaces. (B) Amino acid residues within 3.9 Å of the carotenoid are shown by sticks. (C) Surface-bound watermolecules at the major interface are shown in slate-colored spheres in Synechocystis OCP (PDB ID code 3MG1). This layer of water molecules fully or partiallyeclipses other water molecules, which are either conserved or found to be at the same location (within 0.5 Å) in the crystal structures of A. maxima and Syn-echocystis OCP (Table S6). The coloring indicates their depth from the surface of the OCP as shown in Fig. 4 A and B. Removal of the slate-colored spheres, exposingpartially buried water, is shown in orange inD. The fully buried waters (red spheres) are invisible in the surface diagram of OCP. (E) Cross-sectional view to show theposition of fully and partially buried structural waters in OCPO. (F) Details of water–protein H-bonding network in water cluster 1 at the major interface. Theabsolutely conserved R155 is closely surrounded (<3.2 Å, capable of forming H-bond) by a number of buried (HOH1151,1200, and 1671) water molecules, which areinvolved in dense residue-water interactions as discussed in the main text. Similar H-bonding networks are also observed in the water clusters 2 and 3 (Table S6).



Fig. S2. (A and B) Size-exclusion chromatograms for dark (OCPO) and illuminated OCP samples applied to a Superdex 200 10/300 GL column in different buffers.Experiments were performed in the following buffers: TBS (50 mM Tris·HCl, 200 mM NaCl); PBS (20 mM potassium phosphate, pH 7.4, 100 mM NaCl); 200 mMammonium acetate buffer. Elution volumes for three globular standards are indicated (vertical lines) on the baseline of each chromatogram. Estimated MWs foreach elution peak, as determined from a calibration to the globular standards in each buffer system, are noted on the chromatograms. (C) Circular dichroismspectra of OCPO and OCPR in the far-UV range. The spectra of OCP in darkness (OCPO, black solid line) and after illumination (OCPR, dashed red line) are shown asthe average result of four independent experiments (4 × 8 scans per spectrum). Changes in the average mean-residue-ellipticity values between OCPO and OCPR are

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shown additionally as a difference spectrum (blue dashed line). (D) Far-UV CD difference spectra (OCPR – OCPO) calculated from four independent experiments.Illumination has a small but reproducible effect on the far-UV CD spectra. (E) Time-dependent OCPR CD data collection of OCP samples before and after CDmeasurements. A representative dataset showing successive scans of the OCPR sample (∼2 min per scan) and the lack of significant thermal reversion to the OCPO

form during the full time course of the measurement. An OCPO spectrum (average of eight scans) is also shown for reference. (F) Time-dependent UV-visible(UV-Vis) absorbance spectra of OCP samples before and after CD measurements. UV-Vis spectra collected during the CD experiment: OCPO (black dotted line) wasimmediately illuminated to OCPR (red solid line) after CD measurements on OCPO. The OCPR spectrum after CD acquisition (dotted red line) was acquired after∼20min of spectral averaging (eight scans) at 4 °C in the CD spectrometer. Thermal reversion of OCP after the OCPR measurements resulted in recovery of the OCPO

form (black solid line).

Fig. S3. (A) SEC-SAXS data collection setup for collecting data on OCPR. The eluting OCP from the size exclusion passes the UV detector and is illuminated inthe tubing connecting the UV detector to the capillary, as well as at the first portion of the capillary, above the X-ray beam path. The capillary block wasenclosed in a circulating water bath, held at 8 °C. The LED distance was varied to acquire data for low (20 cm), medium (7 cm), and high illumination (2 cm).OCPO was collected by covering the lines with aluminum foil and blocking all background light near the capillary. An additional control OCP was run bypassingthe UV detector, to test for any background activation of OCP from the UV detector. (B and C) SEC-SAXS traces of OCPO and OCPR. The UV absorbance at 280nm, scattering intensity at zero angle, and radius of gyration are shown for each point throughout the SEC-SAXS run for OCPO (B, kept in the dark) and OCPR

(C, medium LED intensity). (D) SAXS profiles of OCP in the absence of illumination either with (blue) and without (red) online UV detection during SEC-SAXS.(E) The Guinier fits of the merged SEC-SAXS datasets for OCP show good linearity. I(0) and Rg are shown in Table S4.



Fig. S4. Changes in the shape of OCP upon light activation by SAXS. (A) SAXS based ab initio shape reconstruction of OCPO using DAMMIN. The NTD andCTD from the crystal structure are shown in purple and green, respectively (PDB ID code 3MG1). The N-terminal helix is shown in red. (B) Shape reconstructionsof OCPR using DAMMIN. (C) The program Coral was used for rigid body modeling of the two domains to match the SAXS pattern for OCPR. The models showthat, in OCPR, the NTD and CTD are separated by ∼16 Å. The carotenoid ligand is shown in yellow, and the position of R155 is shown in cyan. (D) Models fromthe 20 Coral runs were consistent with the scattering curve (χ = 3.1 ± 0.2), with a χ of 2.59 for the best fit model.



Fig. S5. (A) SEC-MALS: Chromatogram of MALS (red) and differential refractive index (dRI) (blue) from SEC of “zero” and X-ray irradiated OCPO samples usingEthan LC (GE Healthcare Life Science) coupled to Dawn Heleos MALS detector with an embedded dynamics light scattering detector and Optilab rEX dRIdetector (Wyatt Technology Corp.). The data shown are directly from the Astra software. The scattering signal indicates no change in peak characteristics ofthe major component, OCP-monomer (peak “1”) upon X-ray irradiation. The dRI signal indicates presence of buffer components (peak “2” in all of thesamples) and methionine amide (peak “3” in the irradiated samples). (B) Representative XF-MS dose–response plots: peptide modification as a function ofX-ray irradiation dose for residue and residues clusters of OCPO, (black squares) and illuminated OCPR, (red circles). Error bars represent the SE from threeindependent measurements. The solid lines represent single-exponential fits to the dose-dependent data. The ratio of the modification rates (R) indicates thechange in relative accessibilities, which are shown in Table S6.



Fig. S6. (A) Peptic coverage map of OCP. Each observable peptic peptide is shown as a gray bar under the primary sequence of OCP. The figure was made using HXMS tools (www.HXMS.com/mstools). (B) Mass spectra for residues 2–12 after 3 s of deuteration in the orange and red states. Binomial distributions used to fit the mass envelopes are shown in purple. For the OCPR dataset, there is a minor subpopulation of nonactivated OCPO. Based on fitting of two binomialdistributions (orange and blue), this minor population corresponds to ∼4% of the total OCP. (C) Mass spectra for residues 117–133 after 3 s of deuterium exchange in the dark (“orange”), in the presence of blue light, and with 30 s of preillumination (“red”), or in the presence of blue light after 1 h of constant illumination (“post 1 hr illumination”). The identical spectra in the last two panels indicate that even extensive illumination did not disrupt the protein structure. (D)HDX-MS for all observable peptides. The uptake plots for all peptides of OCPO (orange squares) and OCPR (red circles) are shown. The average (mean) percent deuterium exchange is shown relative to the fully deuterated and “zero” standards (SI Methods). Error bars show SD between duplicate measurements. The PPPI internal standard showing identical exchange conditions between the two samples is the last plot at the bottom.


http://www.HXMS.com/mstools


Table S1. Light scattering derived structural parameters

Rh, nm %PdScatteringdetector, V

Molecularmass, kDa*

OCPO 3.252 ± 0.031 6.9 ± 2.4 0.285 ± 0.007 63.5 ± 2*OCPR 3.572 ± 0.024 7.2 ± 2.3 0.287 ± 0.002 65.7 ± 1*

*Because both OCPO and OCPR absorb at a wavelength that overlaps with the laserin the light scattering detector (658 nm), the molecular mass from static scatteringwill be inaccurate (Wyatt Technologies).

Table S2. Fractions of secondary structure content calculated by multiple methods and basis sets for OCPO

and OCPR

Method Basis

OCPo secondary structure content OCPr secondary structure content

Helix Sheet Turn Unrd Helix Strand Turn Unrd

CDSSTR SP29 47.7 ± 1.8 13.4 ± 1.4 17.9 ± 0.8 20.8 ± 0.4 45.5 ± 1.8 15.3 ± 1.2 17.3 ± 0.7 21.8 ± 0.4CDSSTR SP37 47.6 ± 1.5 12.9 ± 1.0 17.3 ± 0.5 22.2 ± 1.1 44.6 ± 1.9 15.9 ± 1.2 16.7 ± 0.5 22.9 ± 0.8CDSSTR SP43 50.7 ± 1.8 11.6 ± 0.9 13.7 ± 0.5 23.9 ± 0.9 47.6 ± 1.8 13.7 ± 0.9 14.8 ± 1.0 24.0 ± 0.6CDSSTR SDP42 47.9 ± 1.7 13.5 ± 1.1 19.2 ± 0.9 19.2 ± 0.9 45.3 ± 1.9 16.8 ± 1.2 19.0 ± 1.0 18.9 ± 0.8CDSSTR SDP48 50.9 ± 1.8 12.0 ± 1.3 15.8 ± 1.5 20.9 ± 0.8 48.0 ± 1.9 15.2 ± 0.9 16.7 ± 0.8 20.0 ± 1.3CONTINLL SP29 46.3 ± 2.0 13.9 ± 1.3 18.7 ± 0.6 21.3 ± 0.6 44.5 ± 1.9 14.9 ± 1.0 17.7 ± 0.9 23.0 ± 0.9CONTINLL SP37 43.9 ± 1.4 14.7 ± 0.9 18.7 ± 0.6 22.8 ± 0.7 40.6 ± 1.1 17.4 ± 0.7 17.2 ± 0.9 24.8 ± 1.1CONTINLL SP43 47.2 ± 1.3 12.2 ± 1.1 16.2 ± 0.2 24.4 ± 0.4 45.6 ± 1.6 13.6 ± 1.1 14.6 ± 0.6 26.2 ± 0.7CONTINLL SDP42 44.1 ± 1.2 15.4 ± 0.9 19.1 ± 0.5 21.5 ± 0.6 41.6 ± 1.2 16.8 ± 0.5 17.9 ± 0.7 23.8 ± 1.2CONTINLL SDP48 47.1 ± 1.4 12.6 ± 1.1 15.5 ± 0.5 24.8 ± 0.3 45.2 ± 1.6 14.6 ± 0.9 14.3 ± 0.6 25.9 ± 0.9SELCON3 SP29 45.3 ± 1.8 13.2 ± 1.0 18.7 ± 0.9 22.8 ± 1.2 43.7 ± 1.7 13.1 ± 0.8 18.2 ± 0.8 24.2 ± 0.7SELCON3 SP37 45.5 ± 1.7 13.3 ± 1.2 17.9 ± 0.8 23.7 ± 0.2 43.2 ± 1.6 13.8 ± 0.9 17.8 ± 1.0 24.8 ± 0.8SELCON3 SP43 47.5 ± 1.6 12.6 ± 1.0 14.6 ± 0.2 25.6 ± 1.6 45.9 ± 1.7 13.1 ± 0.9 13.9 ± 0.5 27.1 ± 1.2SELCON3 SDP42 45.4 ± 1.7 13.8 ± 0.9 18.2 ± 0.7 23.2 ± 0.6 43.4 ± 1.7 13.9 ± 0.8 17.7 ± 1.0 24.5 ± 1.0SELCON3 SDP48 47.5 ± 1.6 12.7 ± 0.9 14.8 ± 0.4 25.4 ± 1.6 45.9 ± 1.7 13.3 ± 0.8 14.1 ± 0.3 26.8 ± 1.1

Average 47.0 ± 1.6 13.2 ± 1.1 17.1 ± 0.6 22.8 ± 0.8 44.7 ± 1.7 14.7 ± 0.9 16.5 ± 0.8 23.9 ± 0.9

Fractions of content are listed as an average result from the analysis of CD spectra from four independent OCPO/OCPR datasets. Thetotal helix and strand content shown here represent the sum of regular and disordered helix/sheet content output by the secondarystructure analysis packages for each of these elements.

Table S3. Differences in secondary structure content in the OCPO andOCPR forms as calculated from the results in Tables S1–S4

Method Basis

Δ(OCPr-OCPo) secondary structure content

ΔHelix ΔStrand ΔTurn ΔUnrd

CDSSTR SP29 −2.3 1.9 −0.6 1.0CDSSTR SP37 −3.0 3.0 −0.6 0.7CDSSTR SP43 −3.1 2.0 1.1 0.1CDSSTR SDP42 −2.6 3.3 −0.2 −0.3CDSSTR SDP48 −2.8 3.2 0.9 −0.9CONTINLL SP29 −1.8 1.0 −1.0 1.7CONTINLL SP37 −3.3 2.7 −1.5 2.0CONTINLL SP43 −1.6 1.4 −1.6 1.8CONTINLL SDP42 −2.5 1.5 −1.2 2.3CONTINLL SDP48 −2.0 2.0 −1.2 1.2SELCON3 SP29 −1.7 −0.1 −0.5 1.5SELCON3 SP37 −2.3 0.5 −0.1 1.2SELCON3 SP43 −1.6 0.5 −0.7 1.5SELCON3 SDP42 −2.0 0.1 −0.5 1.3SELCON3 SDP48 −1.5 0.6 −0.7 1.4

Average -2.2 1.6 -0.6 1.1



Table S4. SAXS derived structural parameters

Rg (Guinier), Å I(0) Peak Abs280 Rg (GNOM), Å Dmax, Å

Dark (no UV) 25.9 ± 0.2 363 ± 2 * * *Dark 25.8 ± 0.2 775 ± 6 0.527 25.7 ± 0.1 82Weak LED 27.6 ± 0.2 813 ± 6 0.576 29.6 ± 0.2 115Medium LED 33.3 ± 0.4 785 ± 8 0.52 37.0 ± 0.3 135High LED 34.5 ± 0.4 937 ± 9 0.574 38.0 ± 0.2 135

*UV was not collected and the SAXS data was collected at a different q range, therefore real space analysis wasnot comparable.

Table S5. Peptides and modification sites detected by XF-MS, and the ratio(s) of hydroxyl radical reactivity for OCPR versus OCPO

Seq. no.* Peptide sequence† Site of modification‡

Ratio “R“ of hydroxyl radicalreactivity kOCP

R/kOCPO§

2–9 PFTIDSAR P2, F3 (+16 Da){ 0.74 ± 0.15A8, R9 (+16 Da) 1.85 ± 0.39

10–27 GIFPNTLAADVVPATIAR Residues 13–22 (+16 Da) 1.93 ± 0.2528–49 FSQLNAEDQLALIWFAYLEMGK W41 (+48 Da) 0.35 ± 0.05

W41, F42, Y44, M47 (+16 Da) 0.47 ± 0.09M47 (+16 Da) 0.76 ± 0.16

50–69 TLTIAAPGAASMQLAENALK A54, A55, P56 (+16 Da) 0.76 ± 0.09M61 (+16 Da) 1.06 ± 0.22

70–89 EIQAMGPLQQTQAMCDLANR M74 (+16 Da) 0.69 ± 0.13M74P76M83 (+16 Da) 0.67 ± 0.02

97–106 TYASWSPNIK Y98W101P103 (+16 and +32 Da) 1.95 ± 0.30107–112 LGFWY F109W110Y111 (+16 and +32 Da) 0.90 ± 0.13113–155 LGELMEQGFVAPIPAGYQLSANANAVLATIQGLESGQQITVLR M117 (+16 Da) 0.69 ± 0.13119–146 QGFVAPIPAGYQLSANANAVLATIQGLE F121,P124,P126 (+16 Da)# 0.17 ± 0.05147–160 SGQQITVLRNAVVD R155, N156 (+16 Da) 10.11 ± 3.96156–167 NAVVDMGFTAGK M161 (+16 Da) 0.68 ± 0.12

F163 (+16 Da) 0.78 ± 0.46K167 (+16 Da) 0.70 ± 0.21

172–185 IAEPVVPPQDTASR P175P178P179R185 (+16 Da) 0.58 ± 0.06192–215 GVTNATVLNYMDNLNANDFDTLIE M202 (+16 Da) 1.1 ± 0.18

Residues 119–215 (+16 Da)# 0.89 ± 0.08221–235 GALQPPFQRPIVGKE Residues 226–231 (+16 Da)# 0.58 ± 0.07243–249 EECQNLK No modification jj

255–268 GVTEPAEDGFTQIK P259, F264, K268 (+16 Da) 1.02 ± 0.11273–289 VQTPWFGGNVGMNIAWR P276, W277, F278 (+32 Da) 2.02 ± 0.23

M284 (+16 Da) 1.54 ± 0.17290–297 FLLNPEGK F190 (+16 Da) 2.16 ± 0.33

L291, L292 (+16 Da) 0.59 ± 0.05P294 (+16 Da) 0.58 ± 0.11

298–310 IFFVAIDLLASPK I298, F299 (+16 Da) 1.21 ± 0.27V301, A302, I303 (+16 Da) 2.08 ± 0.30

P309, K310 (+16 Da) 0.46 ± 0.07

*The 94% sequence coverage was obtained from the bottom-up LC-electrospray ionization (ESI)-MS analysis of OCPO and OCPR using trypsin and Glu-Cdigestion.†Sequences of digested fragments identified by mass spectrometry analysis described in SI Methods.‡Positions of modified residues identified by mass spectrometry analysis described in SI Methods.§The ratio (R) of hydroxyl radical reactivity rate between OCPO and OCPR from three independent measurements. The rate constants (k s−1) were estimated byusing a nonlinear fit of hydroxyl radical modification data to a first order decay as described in SI Methods. R is a quantitative measure of the change in thesolvent accessibility.{Mass shift due to side chain modification is show within the parentheses.#Side chain modification at multiple residues within the specified sequence number.jjNo ratio of hydroxyl radical reactivity.



Table S6. List of water molecules that are either conserved or found to be at the same location (within 0.5 Å) in the crystal structures ofA. maxima and Synechocystis OCP

Water name inPDB ID code3MG1 B Factor

Nearest residue(s)that can form

H bond Distance, ÅCorresponding H2O inPDB ID code 1M98 Location and water cluster (WC)

HOH 1001* 19.6 I303-O† 2.8 HOH451 Minor interface /WC3HOH1042-O 2.7

HOH 1003* 21.4 HOH1042-O 3.8 HOH420 Minor interface /WC3L105-O 3.7L105-N 2.8

HOH 1008* 21.3 R9-NH2 4.0 HOH490 Minor interface /WC3R9-O 2.8

HOH 1019 6.8 ‡ ‡ HOH526 CTDHOH 1020* 16.4 T275-OG1† 2.9 Cl403 Major interface /WC1HOH 1023 8.9 ‡ ‡ HOH500 CTDHOH 1042 22.2 F227-N† 2.9 HOH442 Minor interface /WC3

HOH1001 2.7P225-O† 3.8

HOH1003-O 3.7HOH 1044 19.0 N104-ND2† 3.8 HOH519 Major interface /WC1

N104-N† 3.0HOH1200-O 2.8

E243-O 2.9HOH 1046 17.6 S102-OG§ 2.7 HOH506 Major interface /WC1

E243-O 3.0HOH 1060* 25.1 Y44-OH 2.7 HOH432 NTDHOH 1061 1.0 ‡ ‡ HOH447 CTDHOH 1070 22.5 ‡ ‡ HOH614 NTDHOH 1081 0.1 ‡ ‡ HOH447 CTDHOH 1084 20.9 T15-O† 2.9 HOH663 Minor interface /WC3

D304-OD2† 3.8HOH1395-O 2.7

HOH 1109 34.0 ‡ ‡ HOH699 NTDHOH 1125* 20.0 M284-SD 3.6 HOH410 Major interface /WT2

HOH1342-O 2.7HOH 1128 21.9 N227-NE2 2.7 HOH676 Major interface /WC2HOH 1142 15.9 E311-OE1† 2.8 HO438 Near minor interface /WC3

K268-NZ† 2.8HOH 1147 24.9 Y101-O 2.8 HOH169 Near P103 at the major interface

and water cluster 1HOH 1151 19.1 N104-OD1† 2.9 HOH509 Major interface /WC1

R155-NE† 3.2W277-NE1† 3.7

HOH 1164 22.3 ‡ ‡ HOH558 NTDHOH 1200 18.1 R155-NH2† 3.5 HOH460 Major interface /WC2

N104-OD1† 3.3HOH 1203 18.7 ‡ ‡ HOH408 OthersHOH 1218 16.7 HOH1671-O 2.6 HOH412 Major interface /WC1

HOH1020-O 3.2HOH1200-O 3.6

E244-O 2.9HOH 1259 21.3 HOH1268-O 2.9 HOH512 Near P103 at the major interface

and water cluster 1HOH 1285 36.2 ‡ ‡ HOH640 NTDHOH 1290 21.6 D304-OD1† 2.7 HOH478 Minor interface /WC3

HOH1003-O 2.9HOH1042-O 2.8

HOH 1311 5.8 ‡ ‡ HOH590 Near P179 of the linkerHOH 1313 14.5 E244-O 3.0 HOH473 Major interface /WC1HOH 1317 20.2 ‡ ‡ HOH501 OthersHOH 1342 20.8 HOH1128-O 3.4 Major interface /WC2

HOH1391-O 3.0HOH 1391 23.5 N156-ND2† 3.0 HOH685 Major interface /WC2

N156-OD1† 3.5 HOH474



Table S6. Cont.

Water name inPDB ID code3MG1 B Factor

Nearest residue(s)that can form

H bond Distance, ÅCorresponding H2O inPDB ID code 1M98 Location and water cluster (WC)

HOH 1395 19.1 HOH1084-O 2.7 HOH507 Minor interface /WC3T15-OG1† 3.9I11–O† 3.3

HOH 1448 39.6 ‡ ‡ HOH550 NTDHOH 1467 27.7 N156-ND2† 2.9 HOH695 Major interface /WC2

F227†, P22†

HOH 1491 23.3 ‡ ‡ HOH469 NTDHOH 1610 22.3 N228-OE1 3.0 HOH583 Major interface /WC2HOH 1628 22.3 HOH1259-O 2.9 HOH520 Near P103 at the major interface

and water cluster 1HOH 1664 35.3 ‡ ‡ HOH558 NTDHOH 1671 18.6 R155-NH2† 3.1 HOH629 Major interface /WC1

The nearest neighboring atom on the amino acid residue that can form H bonding with these water molecules, is determined from the crystal structureusing PyMOL v1.2r3pre (Schrödinger, LLC).*Conserved water reported in OCP structures.†Absolutely conserved amino acid residue.‡The nearest residue that can form H bond are not shown, and the distance is not measured. The water molecule is located either at the opposite side of majorinterface or NTD. This water molecule is not linked to the WC, and can play a role in structural stabilization.§Moderately conserved.



supporting information - pnas · supporting information ... analytical sec was performed on an akta...

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