Microviscosity in E. coli Cells from Time-Resolved Linear Dichroism

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Microviscosity in E. coli Cells from TimeResolved Linear Dichroism Measurements Eefei Chen, Raymond M. Esquerra, Philipp A. Meléndez, Sita S. Chandrasekaran, and David S. Kliger J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b07362 • Publication Date (Web): 17 Aug 2018 Downloaded from http://pubs.acs.org on August 21, 2018

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The Journal of Physical Chemistry

Microviscosity in E. coli cells from Time-resolved Linear Dichroism Measurements Eefei Chen†ORCID, Raymond M. Esquerra‡, Philipp A. Meléndez‡, Sita S. Chandrasekaran‡, and David S. Kliger†* †Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064 ‡Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132 ABSTRACT: A protein’s folding or function depends on its mobility through the viscous environment that is defined by the presence of macromolecules throughout the cell. The relevant parameter for this mobility is microviscosity — the viscosity on a time and distance scale that is important for protein folding/function movements. A quasi-null, ultrasensitive time-resolved linear dichroism (TRLD) spectroscopy is proving to be a useful tool for measurements of viscosity on this scale, with previous in vitro studies reporting on the microviscosities of crowded environments mimicked by high concentrations of different macromolecules. This study reports the microviscosity experienced by myoglobin in the E. coli cell’s heterogeneous cytoplasm by using TRLD to measure rotational diffusion times. The results show that photolyzed deoxyMb ensembles randomize through environment-dependent rotational diffusion with a lifetime of 34 ± 6 ns. This value corresponds to a microviscosity of 2.82 ± 0.42 cP, which is consistent with previous reports of cytoplasmic viscosity in E. coli. The results of these TRLD studies in E. coli 1) provide a measurement of myoglobin mobility in the cytoplasm, 2) taken together with in vitro TRLD studies, yield new insights into the nature of the cytoplasmic environment in cells, and 3) demonstrate the feasibility of TRLD as a probe of intracellular viscosity. INTRODUCTION In the cell, large molecules such as nuclei acids, complex sugars, and proteins occupy about 40% of the cell’s total volume. The concentration of proteins ranges from 10% to > 50%, depending on the location in the cell. For example, the concentration in the endoplasmic reticulum is 10-20%, 30% in the red blood cell, and ~70% in the mitochondrial matrix.1-5 To understand the effect of such “crowded conditions”, in vitro studies of protein folding, using macromolecules such as inert sugars (dextran and Ficoll) and proteins (bovine serum albumin (BSA) or lysozyme), are used to mimic cellular conditions. These studies have shed light on how crowding factors (excluded volume, viscosity, electrostatics…) can alter the kinetic mechanisms of protein folding significantly, as well as appreciably enhance the formation of aggregates, such as those implicated in neurodegenerative diseases.6-8 Although excluded volume is considered the most significant consequence of crowding at high macromolecular concentrations, the protein’s ability to function or to fold also relies on its mobility through the heterogeneous, viscous environment that is defined by the presence of macromolecules throughout the cell. The relevant parameter for this mobility is microviscosity — the viscosity on a distance scale that is important for the movements involved in protein folding/function. Microviscosity differs from macroviscosity (or bulk viscosity), which is determined by the diffusion of an infinitely large probe using viscometers or rheometers. In contrast, microviscosity is determined by methods that can measure rotational or translational diffusion over nanometer to micrometer spatial scales. Common techniques for microviscosity measurements

include fluorescence or absorption anisotropy, fluorescence correlation (FCS), lifetime imaging (FLIM), recovery after photobleaching (FRAP), and microphotolysis (FM) spectroscopy, NMR and EPR methods.9-21 Recently, this laboratory used time-resolved linear dichroism (TRLD) spectroscopy to obtain microviscosities for solutions of a variety of macromolecular crowders ranging in concentration from 0 to 500 mg/mL.22 These solution conditions are often used in studies of protein folding mechanisms to understand the effect of crowded environments. The in vitro TRLD results were considered in the context of cellular viscosity studies to qualitatively determine their relevance to intracellular environments. For example, in the dense environment of rat liver mitochondria (560 mg/mL protein concentration) fluorescence anisotropy studies report viscosities that are 37 times more viscous than water.4, 23 Chen et al. show that a solution of BSA at pH 6.5 has a microviscosity of 37 cP with a calculated protein concentration of 578 mg/mL, which is very close to the measured 560 mg/mL.22 When other macromolecules (dextran and Ficoll) are considered, a viscosity of 37 cP corresponds to calculated concentrations of 490 to 630 mg/mL, averaging around 560 mg/mL. Because of the cellular complexities that lead to difficulties in interpretation of in vivo data,24 in vitro studies can be very important in understanding what factors control protein folding and function in cells. That is, it is possible to closely and independently control artificially crowded environmental factors, such as macromolecular size and interactions, concentration, pH, and viscosity. Carrying out measurements in cells is important, however, to provide a check on the relevance of in

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vitro measurements. Previously reported in vitro TRLD studies by this laboratory probed rotational diffusion of myoglobin as a function of different macromolecule characteristics - molecular weights, sizes, shapes, and surface charges.22 Here, TRLD is used to measure the rotational diffusion time of MbCO expressed in E. coli (denoted cMbCO) within the cell. Using the simple and empirically determined functions from Chen et al.,22 the resulting TRLD data indicate that the microviscosity of the cytoplasm is only about 3 times more viscous than water. The results of these in vivo TRLD studies 1) provide a measurement of myoglobin mobility in the cytoplasm, 2) taken together with in vitro TRLD studies, yield new insights into the nature of the cytoplasmic environment in cells, and 3) demonstrate the feasibility of TRLD as a probe of intracellular viscosity.

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Nanosecond Polarization Spectroscopy. Nanosecond polarization methods used in this laboratory are based on a quasinull optical approach. With TRLD measurements, the quasinull method is based on nearly-crossed linear polarizers – that is, one polarizer in the probe beam is rotated clockwise and then counterclockwise by a small angle, β, off their initially crossed (at -45° and +45° from the vertical polarization of the laser) orientations. To a good approximation, the pure LD signal (S ≈ LD/β) is magnified when β is much less than 0.017 radians.27 Likewise, for time-resolved optical rotatory dispersion (TRORD) measurements, one polarizer is rotated first by -β and then by +β from the initially crossed positions of 0 and 90° from vertical. ORD is the rotation of the plane of linearly polarized light by circular birefringence (CB) from an optically active sample. The signal then is S ≈ -CB/β. More often than not, the conditions are not ideal because of imprecise alignment of polarizers, large β (in this case 0.17 radians), or an imperfect polarizer extinction ratio. Not only do these factors attenuate the signal, but they will also introduce either CB or LD to the respective TRLD or TRORD measurement. That is, the signal becomes S ≈ (LD-CB)/β. TRLD (rotational lifetime) measurements. The ultrasensitive TRLD method used in this study has been described in detail by Che et al.27 Briefly, a Quanta Ray (Spectra Physics, Santa Clara, CA) DCR-2A Nd:YAG laser (7 ns FWHM, 22-23 mJ, 532 nm) was used to photolyze CO from cMbCO or bMbCO. Because the laser pulses are linearly polarized, photolysis photoselects an oriented deoxyMb subpopulation, resulting in a linearly dichroic sample.10 Over time, the photoselected protein molecules randomize their orientations and the LD decays. MbCO is an ideal protein for these in vivo studies for several reasons. 1) The reorientation through rotational diffusion occurs on a time and distance scale that is on the order of protein movements expected in folding/function reactions. 2) As shown in Figure 4A of Chen et al.,28 the rotational diffusion measurement is not compromised by absorption changes that follow photolysis of CO and its subsequent recombination. And 3), in vitro TRLD measurements of bMbCO have been demonstrated to be an effective probe of microviscosity in crowded environments.22 The changes that follow photolysis of cMbCO or bMbCO were monitored with an apparatus using a 2 µs xenon flash lamp probe source and two linear polarizers oriented at +45o and 45o from the vertical polarization of the laser. After the monitoring beam passes through these elements, the probe light is directed into a spectrograph and an intensified CCD detector. LD is obtained when the probe beam intensity is measured first with the analyzing polarizer rotated by a small angle of β and then again with that polarizer at –β. The LD signal is calculated from the normalized difference of the intensities measured at ±β. That is, signal = [(I+β – I-β)/(I+β + I -β)] ≈ LD/β. In this study, β = 0.17 radians. Light scattering due to the E. coli cells was minimized by index matching with glycerol. All cMbCO samples measured contained 10% glycerol. The resulting rotation of the plane of polarized probe light due to CB of glycerol was counterrotated by measuring TRLD with the crossed position of the two polarizers determined in the presence of the sample. TRLD data for cMbCO were measured at 10 time points (40, 80, 130, 230, and 530 ns, 1, 10, and 500 µs, and 1 and 10 ms)

METHODS Materials. Horse skeletal myoglobin (metMb) (SigmaAldrich, St. Louis, MO), monobasic and dibasic sodium phosphate (NaP) and glycerol (Thermo Fisher Scientific, Waltham, MA), and sodium hydrosulfite (Fluka Chemicals, Ronkonkoma, NY) were used without further purification. Sample Preparation. For time-resolved experiments, MbCO in buffer (denoted bMbCO) was prepared by adding enough metMb to 50 mM NaP (pH 7) for a concentration of 7-8 µM. Carbon monoxide was flowed through the metMb sample for approximately 0.5 h before sodium hydrosulfite was added. The final bMbCO solution was sealed in an airtight 5 mm quartz cuvette for all experiments. BL21 (DE3) competent cells expressing wild-type sperm whale myoglobin in a pMb221 plasmid were transformed and expressed following established protocols.25 One gram of harvested cell pellet was resuspended in about 1 mL of 50% glycerol. The resuspended cells were diluted with 100 mM NaP buffer, pH 7.4, to achieve 10% glycerol. Samples were transferred to a 1 x 0.5 cm quartz cuvette and placed under 1 atm of CO for 40 minutes, and then anaerobically sealed. Sample Integrity Metrics. The concentrations of metMb and MbCO were calculated using extinction coefficients ε408 = 188,000 and ε424 = 207,000 M-1 cm-1, respectively.26 Absorbance values for these determinations were obtained from spectra measured on a UV-Vis spectrophotometer (V750, JASCO, Inc., Easton, MD). Spectra were measured for each sample before, during, and after each experiment. Refractive index values were measured on a refractometer (ABBE-3L, Milton Roy, Raleigh, NC) to determine the concentration of glycerol added to the cMbCO solutions. The refractive index of each sample was checked before the start of measurements, during, and then after all TRLD experiments were complete. Intracellular versus Extracellular MbCO. To confirm that TRLD measurements were performed on cMbCO inside E. coli rather than protein that had escaped the cells, the samples were pelleted in a microcentrifuge (Microfuge E, Beckman Instruments, Palo Alto, CA). A UV-Vis spectrum of the supernatant was measured to check for absorption features characteristic of either metMb or MbCO. In all reported cases, the presence of free Mb from lysed cells was not detectable.

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after photolysis of the CO ligand. A total of 8,600 (43 x 200) averages were obtained from 4 different cMbCO preparations. A 340 nm cutoff filter (5-61, Corning Glass Works, Corning, NY) was used to avoid spectral effects due to second-order light. For bMbCO, 200 (4 x 50) averages at each of 18 time delays between 2.5 ns and 10 ms were measured. The samples were maintained at 21 °C during the experiments. Time-resolved optical rotatory dispersion (TRORD) measurements. TRORD measurements on cMbCO and bMbCO were performed on the same apparatus used for TRLD experiments, except that the two linear polarizers were crossed at 0º and 90º rather than at +45º and -45º from vertical polarization. Vertically polarized laser light was used to initiate the photolysis reaction. For details of the TRORD system see Shapiro et al.29 As with the LD measurement, the CB signal (or ORD spectrum) is determined from S = [(I+β – I-β)/(I+β + I-β)] ≈ CB/β, where β for these experiments was also 0.17 radians. I+β and I -β are the probe light intensities when the analyzing polarizer is rotated by +β and -β. The TRORD data were measured at the same time delays collected for the TRLD data. A total of 1400 (7 x 200) averages and 256 (4 x 64) averages were measured for cMbCO and bMbCO, respectively. Time-resolved absorption (TROD) measurements. TROD data were measured on the same time-resolved configuration that was used to measure LD and ORD. The only difference between the two configurations is that for TROD the probe beam was depolarized. The TROD data 224 (7 x 32) averages were obtained at 16 time delays between 40 ns and 10 ms. Data Analysis. The TRLD, TRORD, and TROD data were analyzed using singular value decomposition (SVD) and global kinetic fitting algorithms written in Matlab (The MathWorks, Inc., South Natick, MA). These mathematical approaches have been described previously.30

Figure 1. A) TRLD measurements of cMbCO at 10 time delays after photolysis of cMbCO. The earliest, 40 ns (black line and dots), and latest, 10 ms (black line), times are highlighted. Data comprising 8,600 averages were best fit to two exponential processes with lifetimes (amplitudes) of 34 ns (0.66) and 1.1 ms (0.27). B) b-spectra (decay-associated spectra) corresponding to these two processes (34 ns (black line and dots) and 1.1 ms (grey line)) are shown. b0 is the spectrum at infinite time (grey line)

The assignment of the 34 ns process to rotational diffusion is based on three factors. First, the results of bMbCO TRLD measurements (Figure 3) report two exponential processes with lifetimes of 9.62 ± 0.36 ns (0.66 ± 0.07) and 1.61 ± 0.08 ms (0.29 ± 0.08). This is consistent with previous TRLD studies of MbCO in buffer which have assigned the 10 ns process to rotational diffusion.22, 28 The longer, 34 ns, lifetime reflects the different rotational diffusion times in cellular versus buffer environments.

RESULTS Figure 1A shows representative TRLD measurements on cMbCO. The data were measured at 10 time delays from 40 ns to 10 ms after photolysis of the heme-CO ligand. The spectral behavior of time dependent LD data comprising 8,600 averages is best fit to two exponential components with lifetimes of 34 ± 6 ns and 1.1 ± 0.1 ms and respective amplitudes of 0.66 ± 0.04 and 0.27 ± 0.03. The b-spectra (or decay-associated spectra) corresponding to these two components are shown in Figure 1B. The nanosecond process is assigned to rotational diffusion (τ=34 ns) of deoxyMb and the millisecond component to an ORD contribution associated with CO recombination (τ=1.1 ms) to reform MbCO (see below). TROD experiments on cMbCO samples show that the nanosecond TRLD signal is due to changes in LD (and thus rotational diffusion) rather than to CO religation kinetics. SVD and global kinetic analysis of the TROD data (Figure 2) yield two exponential processes with lifetimes of 609 ± 167 ns and 1.23 ± 0.07 ms and respective amplitudes of 0.084 ± 0.006 and 0.88 ± 0.02. The cMbCO TROD lifetimes and amplitudes are consistent with the previously reported geminate recombination and bimolecular recombination phases (see Table 1).31 The earliest TROD process (τ = 609 ns) is considerably longer and smaller in amplitude than detected in the TRLD experiments (τ = 34 ± 6 ns (0.66 ± 0.04)).

Figure 2. Results of TROD measurements of cMbCO photolysis. These spectra represent 224 averages measured at 16 times points from 40 ns to 10 ms following sample photolysis. These data are best fit to a fast 609 ns and a slow 1.23 ms process with corresponding amplitudes of 0.084 and 0.88.

Second, because of the small signal size and the significant sample scattering, the rotation angle β used in these TRLD studies was relatively large compared to that typically used for measurements of proteins in buffer. As β increases, simultaneous attenuation and enhancement of the LD and ORD, respec-

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tively, is expected. The TRLD signal is then no longer approximated by S ≈ LD/β, but becomes S ≈ (LD-CB)/β. To test how the TRORD contribution might affect the TRLD measurements, TRORD data were collected for both bMbCO and cMbCO. For bMbCO, the data can be fit to two lifetimes (398 ± 67 ns (0.052 ± 0.001) and 1.53 ± 0.05 ms (0.89 ± 0.01)).

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TRORD, and TROD experiments on cMbCO and bMbCO are summarized in Table 1.

Figure 3. TRLD results measured after photolysis of bMbCO. The data comprise 200 averages measured at 18 times points from 2.5 ns to 10 ms. These data are best fit to a fast 9.6 ns and a slow 1.6 ms process with corresponding amplitudes of 0.66 and 0.29.

Figure 4. Comparison of b-spectra from TRLD, TRLDHP,and TRORD results for bMbCO and cMbCO. A) The b-spectra for the slow, millisecond component from TRLD and TRLDHP studies of cMbCO (1.1 and 1.6 ms, respectively) and TRORD and TRLDHP experiments on bMbCO (1.53 and 1.68 ms, respectively) are similar. These b-spectra have been normalized and overlaid, with the spectrum for the 1.1 ms TRLD cMbCO process also multiplied by -1. B) The resultant b-spectrum for the 34 ns process obtained from analysis of TRLD measurements on cMbCO is spectrally similar to that for bMbCO (10 ns), but distinct from those reported by the analysis of TRORD and TRLDHP data for bMbCO (398 ns and 288 ns, respectively). The b-spectra for the TRORD and TRLDHP processes (for bMbCO) are multiplied by 5 to facilitate spectral comparison. For the 10 ns TRLD bMbCO component, the b-spectrum is divided by 14. TRLDHP measurements on cMbCO could only be fit to a single, millisecond exponential process (1.6 ms). The black lines represent the b-spectra generated from the cMbCO TRLD data analysis and for all other experiments the bspectra are shown in grey.

As expected, these lifetimes are similar to those reported by TROD experiments and are assigned to nanosecond geminate recombination of CO to deoxyMb and to a slower component that reflects bimolecular CO recombination. The lifetimes measured for TRORD of cMbCO (651 ± 140 ns (0.25 ± 0.02) and 2.0 ± 0.9 ms (0.54 ± 0.03)) are similarly assigned to the geminate recombination process and the slower, millisecond CO rebinding phase. (This work also shows the feasibility of time-resolved ORD measurements in vivo.) Third, measuring rotational diffusion with the TRLD method depends on the use of vertically polarized laser pulses to photoselect oriented deoxyMb. If the 34 ns process is indeed attributable to rotational diffusion, then the use of horizontally polarized laser excitation should significantly attenuate or eliminate this process. Thus, using horizontally polarized 532 nm laser pulses, the TRLD measurements (TRLDHP) were repeated on cMbCO (700 averages), as well as on bMbCO (192 averages). SVD and global kinetic analysis can fit the TRLDHP data on bMbCO to two exponential lifetimes – 288 ± 52 ns (0.09 ± 0.03) and 1.68 ± 0.02 ms (0.88 ± 0.02). For TRLDHP experiments on cMbCO only a single exponential with a lifetime of 1.6 ± 0.8 ms (0.7 ± 0.1) could be fit to the data. The resulting b-spectra are compared in Figure 4. Figure 4A shows that the b-spectra for the slow millisecond process from these control TRORD and TRLDHP experiments can be overlaid with that from the TRLD data. This comparison suggests that the b-spectrum corresponding to the second exponential process from the TRLD measurements (τ = 1.1 ms) on cMbCO is an ORD spectrum that can be assigned to CO recombination. The b-spectrum for the 34 ns component of the TRLD studies on cMbCO is shown in Figure 4B with the nanosecond processes reported by the TRORD and TRLDHP data on bMbCO. This comparison, together with the absence of the fast process in the null TRLD experiment on cMbCO, where the reaction is triggered by horizontally polarized laser light, supports the assignment of the 34 ns process to rotational diffusion. The results of the TRLD, TRLDHP,

To address whether the experiments truly measured intracellular MbCO TRLD signals, rather than MbCO that might have leaked from cells, the samples were centrifuged and UV-Vis spectra were measured on the supernatant. Figure 5 shows the UV-Vis spectrum for a typical cMbCO sample and the averaged spectrum of the supernatant from several samples after the cMbCO was pelleted. This examination of the supernatant confirms that the TRLD measurements were on intracellular MbCO. That studies of E. coli cells which do not contain MbCO showed no LD signals demonstrates that the TRLD spectra were indeed a result of cMbCO photolysis. Finally, the possibility of glycerol transport into the E. coli cells was first considered in terms of the most extreme situations. If somehow the cytoplasm was entirely displaced by 10% glycerol, then the rotational diffusion time would have been 15 ns and the microviscosity 1.2 cP. On the other hand, if 2.8 cP is attributable entirely to glycerol then the internal glycerol concentration would be 37%, which is unlikely because the cells were suspended in 10% glycerol. These numbers are based on previous bMbCO TRLD studies.22 For situations in between these extremes, consider the glycerol uptake mechanism. Ex-

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ternal glycerol traverses non-selectively across the outer membrane via porins and then selectively through the inner membrane via aquaglyceroporins.32 Glycerol is trapped in the cell because of phosphorylation to glycerol-3-phosphate by glycerol kinase, but is then further metabolized. 33 This phosphorylation is the basis of the constant external and internal glycerol imbalance and is the “driving force for the uptake of glycerol”.34 Thus, with uptake and metabolism, accumulation of external glycerol inside E. coli is not expected. Still, using the cellular concentration of glycerol-3-phosphate, it is possible to gauge the maximum level of glycerol that may be in the cell at a given time. The average concentration of glycerol-3phosphate measured from E. coli cells grown in 4 g/L glycerol was reported to be about 813 µM.35 This concentration would be less than 1% in terms of glycerol in the cytoplasm and would not contribute significantly to the measured in vivo microviscosity of 2.8 cP.

Table 1. Results of TRLD, TROD, & TRORD experiments Method

Sample

τ1 (ns)

τ2 (ms)

TRLD

cMbCO

34 ± 6 (0.66 ± 0.04)

1.1 ± 0.1 (0.27 ± 0.03)

9.62 ± 0.36

1.61 ± 0.08

(0.66 ± 0.07)

(0.29 ± 0.08)

cMbCO

609 ± 167 (0.084 ± 0.006)

1.23 ± 0.07 (0.88 ± 0.02)

bMbCO31

370 ± 30 (0.07 ± 0.02)

1.3 ± 0.04 (0.94 ± 0.1)

cMbCO

---

1.6 ± 0.8 (0.7 ± 0.1)

288 ± 52

1.68 ± 0.02

(0.09 ± 0.03)

(0.88 ± 0.02)

651 ± 140

2.0 ± 0.9

(0.25 ± 0.02)

(0.54 ± 0.03)

398 ± 67 (0.052 ± 0.001)

1.53 ± 0.05 (0.89 ± 0.01)

bMbCO TROD

TRLDHP

bMbCO TRORD

cMbCO bMbCO

Discussion of viscosities relative to these protein movements requires a metric on a similar, nanometer length scale (microviscosity). Unlike macroviscosity (or bulk viscosity), the measurement of microviscosity requires a different approach, one that can monitor either rotational or translational diffusion. In the cell, diffusion of exogenous and endogenous fluorophores and imported labels or magnetic particles have been coupled with fluorescence methods such as anisotropy, FCS, FRAP, FLIM, and FM, NMR, ESR, and absorption anisotropy.38-57 For reviews see references 48, 56, and 57. This TRLD study follows the decay of anisotropy that is induced by photoexcitation of bMbCO and cMbCO with linearly polarized laser light. The photoselected, anisotropic deoxyMb ensemble randomizes through environment-dependent rotational diffusion. Unlike the classical approach that separately measures the parallel and perpendicular transient absorption signals,58 the pseudo-null, ultrasensitive measurement used here27 enhances the signal size and the signal-to-noise ratio of a linear dichroism signal. TRLD measurements are successful when the diffusional processes of interest are sufficiently separated in time from the chemistry of the reaction. In the case of bMbCO, the rotational diffusion time constant of ca. 10 ns is 20 times faster than the ca. 200-400 ns geminate recombination process.31, 59-60 The nanosecond rotational diffusion time (τ) reported by TRLD studies was used to calculate viscosity on the same molecular distance scale (or microviscosity, µη) using the Stokes-Einstein-Debye equation, µη = 3kTτ / (4πRh3), where µη is substituted for (macro)viscosity, k is the Boltzmann constant, T is temperature, and Rh is the hydrodynamic radius of Mb. With the goal of 1) measuring the viscosity of cytoplasm in E. coli cells and 2) reconciling in vivo results with those measured in vitro, the outcome of the TRLD measurements on cMbCO are compared to those in vitro studies on bMbCO that correlate microviscosity, macromolecular concentration, and rotational diffusion lifetimes.22

Figure 5. UV-visible spectra of cMbCO and the supernatant from a pelleted cMbCO sample. The supernatant (grey) shows no significant absorbance from metMb or MbCO, as indicated by the lines at 408 and 424 nm. In contrast, a typical TRLD cellular sample has a spectrum that is characteristic of MbCO (black), as well as substantial scattering. The inset shows that the Mb sample before CO is introduced comprises a mixture of metMb and oxygen-bound Mb, resulting in an absorbance maximum of 413 nm.

DISCUSSION Macromolecules are the fundamental units of the architecture that govern, through both concentrations and physical attributes, the microscopic spatial diversities in the cell. These heterogeneities give rise to two major crowding factors — excluded volume and viscosity. The latter is important to the diffusional motions that are required to achieve successful biological events, such as protein folding and function, protein translocation, protein-protein interactions and protein-ligand binding. It has been suggested that when a particle (in this case protein amino acids/domains) travel small distances, encountering few macromolecules, its diffusion will be much like that expected in water. In contrast, when the diffusion distance is longer, with increased chances of encounters with large molecules, the diffusion constant has been shown to be smaller than that for water.36-37

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Control studies. As described in the Results section, several control experiments were performed to demonstrate that the 34 ns component of the TRLD measurements is due to rotational diffusion. TROD measurements showed that the TRLD decay is not due to CO recombination and TRORD measurements showed that the long lifetime obtained in the TRLD experiments was actually due to ORD rather than LD. The fact that the 34 ns lifetime is due to LD decay is also shown by TRLD measurements using horizontal laser polarization for sample excitation. To verify that rotational diffusion experiments measured MbCO inside the cells rather than MbCO that may have escaped from the cells into the glycerol/water solution in which the cells were suspended, UV-Vis measurements of supernatant from centrifuged MbCO samples were performed. As with the supernatant experiments, TRLD studies on samples that comprised only cells and no MbCO did not show LD features, confirming that measurements of cMbCO can be attributed to rotational diffusion of the protein. Microviscosity from rotational diffusion lifetime. The 34 ns rotational diffusion time for cMbCO extrapolates to an environment containing about 20 ± 4 % (w/v) macromolecules. This percentage is an average of protein and inert macromolecule concentrations (ranging from 16 to 24%) calculated from in vitro TRLD measurements on bMbCO in the presence of dextrans, Ficoll, and BSA.22 It is consistent with the reported ~30% (w/w) (or ~30% (w/v) or 200-320 mg/mL) relative to the water content of the E. coli cell.61-62 From the 34 ns rotational diffusion time and the calculated 20% w/v macromolecule concentration, the microviscosity in the cytoplasm of E. coli cells is estimated to be 2.82 ± 0.42 cP. This qualitative comparison suggests that the rotations of deoxyMb after photolysis of cMbCO in the E. coli cell are not impeded significantly compared to equivalent movements in water. The results of these TRLD measurements are similar to those reported from 19F NMR spectroscopy, where the contribution of viscosity to rotational diffusion of two globular proteins labeled with analogs of tryptophan and tyrosine was probed also in E. coli cells.63 These in-cell studies estimate that the viscosity of the E. coli cytoplasm is only 2-3 times that of water. Many other studies that probe rotational diffusion also report similar cytoplasmic viscosities (1.1-3.6 cP), but these experiments were probed in different cells, such as yeast Saccharomyces cerevisiae, hamster lung and J774 mouse macrophage cells, CHO cells, Swiss 3T3 fibroblasts, SiHa and Ect1 cell lines, erythrocytes, and sea urchin eggs.39, 41, 43, 51-54, 64-69 In contrast, the results of FRAP and FM show considerably more variability of viscosities (3.7-14 cP) in the cytoplasm of Swiss 3T3 fibroblasts, SK-OV-3, E. coli, and hepatoma tissue cells.43, 46, 49, 70-72 Unlike the viscosities obtained from rotational diffusion in cells, studies that calculate viscosities through translational diffusion measurements generally report a larger value. Of course there are exceptions, such as the 140 cP viscosity of SK-OV-3 cell vesicles measured by FLIM studies using molecular rotors, which reports on intramolecular twisting motions.73 Several reasons are possible for the range of viscosities from different studies. Probe size can play a factor in diffusional differences, but for the most part the molecules in the studies mentioned above were within the weight and size limits of 114 kDa and < 17 nm where the mobility can be de-

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scribed by the Einstein-Stokes prediction and should similarly diffuse through the cytoplasm of E. coli.74-75 Differences in viscosities could also arise from the possibility of probe binding to different parts of the cell or probing during different times of the cell cycle.57 More likely, however, the variation in viscosity is due to both the location of the probe and the increased probability of collisional interactions during translation versus rotation. In the former, the 140 cP calculated by FLIM studies of SK-OV-3 cells suggests that the molecular rotor is localized to an environment that is significantly different from water.73 As an example of the latter, FRAP measurements monitor the recovery of a photobleached area due to translational diffusion of a probe molecule from the unbleached area of the cell. FRAP studies in E. coli DH5α cells, showed that GFP (27.5 kDa) diffusion was about 11 times slower (7.7 µm2/s) than in water (87 µm2/s)66 when it traveled the 3-5.5 µm length of the cells.46 This extrapolates to a viscosity of about 11 cP, which is about 4 times more viscous than the 2.82 cP calculated from these TRLD rotational diffusion measurements. Together, the above comparisons underscore the scale dependence of viscosity, with heterogeneity of the cytoplasm being increasingly realized as the probe traverses the distance of the cell rather than rotating around nanometer lengths in a localized region. That is, while differences in probe characteristics, cell type and cycle, or intracellular location may contribute to viscosity, so does the length scale that is probed. Indeed, studies of water dynamics in the cytoplasm of E. coli have suggested that as the distance scale approaches the atomic level, the viscosity becomes like that of pure water.76 In the context of proteins/peptides, the earliest events of folding can be described with nanosecond time and nanometer distance scales. Helix nucleation reportedly can be as fast as 0.1-1ns and have an upper limit of about 1 µs.77-78 For a short alanine-based peptide, computational analysis of temperature jump experiments indicated it takes about 315 ns to form one helical turn and 5.9 ns to elongate that helix nucleus by one residue.79 Assuming a rigid linear primary sequence (“unfolded”), this means that residues roughly 1.5 nm (maximum distance calculated based on bond lengths of Cα-C, C-N, and N-Cα) apart must come to within 0.54 nm to form the first helical turn with a hydrogen bond between O(i) and N(i+4). Formation of intrachain contacts for 4-30-residue peptides occurs on the early nanosecond time scale (5 to 34 ns, respectively).80 In the presence of Ficoll70 it was demonstrated that intrachain diffusion for those 4-30-residue peptides remained fast despite impeded translational diffusion. Thus, the distance dependence of viscosity suggests that on this nanosecond scale early protein folding events in the cell should experience an environment similar to buffer conditions. This idea may explain why the burst phase (~270 ns) folding of reduced cytochrome c in buffer and in 310 mg/mL sucrose (8 cP) is similar.28 CONCLUSION In conclusion, knowing that the cell is crowded with sugars, nucleic acids, and protein macromolecules raised the obvious question of how closely the results of biophysical studies on proteins in buffer solutions report on intracellular function/folding processes. To address this question, in vitro studies introduced macromolecules to buffer solutions to mimic

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ACKNOWLEDGMENT

intracellular environments and biophysical technology made advances to probe proteins in vivo. In the latter, the presence of these macromolecules, as well as other factors, such as timing of cell cycles, osmolytes, electrostatics, and pH, introduced complexities that make it difficult to directly measure specific biochemical processes and, especially, to interpret the results of these studies. As these two opposing issues (intracellular mimics versus in vivo probes) converge, it is becoming clear that one needs the other to facilitate complete understanding of protein folding and function in the cell. That is, while it may be ideal to measure biochemical processes directly in the cell, it is more difficult to understand the basics of these cellular processes when considering influencing factors such as pH, charge, temperature, viscosity and more. The studies presented here hope to demonstrate the interdependence of in vitro and in vivo studies in an effort to reveal important principles in intracellular protein folding and dynamics.

We thank Robert Goldbeck for helpful discussions and suggestions. This work was funded by NIH MARC T34-GM008574 (SC; PM), The Arnold and Mabel Beckman Foundation’s Beckman Scholars Program (SC), and NSF STC Center for Cellular Construction 1548297 (RE).

ABBREVIATIONS Linear dichroism, LD; time-resolved linear dichroism, TRLD; circular birefringence, CB; optical rotatory dispersion, ORD; time-resolved optical rotatory dispersion, TRORD; myoglobin, Mb; carbonmonoxymyoglobin, MbCO; singular value decomposition, SVD.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]. Phone: 831.459.2106. Fax: 831.459.2935. ORCID Eefei Chen: 0000-0001-7411-7779 The authors declare no competing financial interest. 14. Suhling, K.; French, P. M.; Phillips, D. Time-resolved fluorescence microscopy. Photochem. Photobiol. Sci. 2005, 4, 13-22. 15. Verkman, A. S. Diffusion in cells measured by fluorescence recovery after photobleaching. Methods Enzymol. 2003, 360, 635-648. 16. Reits, E. A.; Neefjes, J. J. From fixed to FRAP: Measuring protein mobility and activity in living cells. Nat. Cell Biol. 2001, 3, E145147. 17. Peters, R. Fluorescence microphotolysis. Diffusion measurements in single cells. Naturwissenschaften 1983, 70, 294-302. 18. Keith, A. D.; Snipes, W. Viscosity of cellular protoplasm. Science 1974, 183, 666-668. 19. Keith, A. D.; Snipes, W.; Mehlhorn, R. J.; Gunter, T. Factors restricting diffusion of water-soluble spin labels. Biophys. J. 1977, 19, 205-218. 20. Serber, Z.; Keatinge-Clay, A. T.; Ledwidge, R.; Kelly, A. E.; Miller, S. M.; Dotsch, V. High-resolution macromolecular NMR spectroscopy inside living cells. J. Am. Chem. Soc. 2001, 123, 2446-2447. 21. Serber, Z.; Ledwidge, R.; Miller, S. M.; Dotsch, V. Evaluation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectroscopy. J. Am. Chem. Soc. 2001, 123, 8895-8901. 22. Chen, E.; Kliger, D. S. Time-resolved linear dichroism measurements of carbonmonoxy myoglobin as a probe of the microviscosity in crowded environments. J. Phys. Chem. B 2017, 121, 7064-7074. 23. Scalettar, B. A.; Abney, J. R.; Hackenbrock, C. R. Dynamics, structure, and function are coupled in the mitochondrial matrix. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 8057-8061. 24. Hingorani, K. S.; Gierasch, L. M. Comparing protein folding in vitro and in vivo: Foldability meets the fitness challenge. Curr. Opin. Struct. Biol. 2014, 24, 81-90. 25. Esquerra, R. M.; Jensen, R. A.; Bhaskaran, S.; Pillsbury, M. L.; Mendoza, J. L.; Lintner, B. W.; Kliger, D. S.; Goldbeck, R. A. The pH dependence of heme pocket hydration and ligand rebinding kinetics in photodissociated carbonmonoxymyoglobin. J. Biol. Chem. 2008, 283, 14165-14175. 26. Antonini, E.; Brunori, M. Hemoglobin and myoglobin in their reactions with ligands. North-Holland Publishing Company: The Netherlands, 1971.

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Figure 1. A) TRLD measurements of cMbCO at 10 time delays after photolysis of cMbCO. The earliest, 40 ns (black line and dots), and latest, 10 ms (black line), times are highlighted. Data comprising 8,600 averages were best fit to two exponential processes with lifetimes (amplitudes) of 34 ns (0.66) and 1.1 ms (0.27). B) b-spectra (decay-associated spectra) corresponding to these two processes (34 ns (black line and dots) and 1.1 ms (grey line)) are shown. b0 is the spectrum at infinite time (grey line). 93x119mm (300 x 300 DPI)



Figure 2. Results of TROD measurements of cMbCO photolysis. These spectra represent 224 averages measured at 16 times points from 40 ns to 10 ms following sample photolysis. These data are best fit to a fast 609 ns and a slow 1.23 ms process with corresponding amplitudes of 0.084 and 0.88. 85x70mm (300 x 300 DPI)


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Figure 3. TRLD results measured after photolysis of bMbCO. The data comprise 200 averages measured at 18 times points from 2.5 ns to 10 ms. These data are best fit to a fast 9.6 ns and a slow 1.6 ms process with corresponding amplitudes of 0.66 and 0.29. 83x63mm (300 x 300 DPI)



Figure 4. Comparison of b-spectra from TRLD, TRLD¬HP,and TRORD results for bMbCO and cMbCO. A) The b-spectra for the slow, millisecond component from TRLD and TRLDHP studies of cMbCO (1.1 and 1.6 ms, respectively) and TRORD and TRLDHP experiments on bMbCO (1.53 and 1.68 ms, respectively) are similar. These b-spectra have been normalized and overlaid, with the spectrum for the 1.1 ms TRLD cMbCO process also multiplied by -1. B) The resultant b-spectrum for the 34 ns process obtained from analysis of TRLD measurements on cMbCO is spectrally similar to that for bMbCO (10 ns), but distinct from those reported by the analysis of TRORD and TRLDHP data for bMbCO (398 ns and 288 ns, respectively). The b-spectra for the TRORD and TRLDHP processes (for bMbCO) are multiplied by 5 to facilitate spectral comparison. For the 10 ns TRLD bMbCO component, the b-spectrum is divided by 14. TRLDHP measurements on cMbCO could only be fit to a single, millisecond exponential process (1.6 ms). The black lines represent the b-spectra generated from the cMbCO TRLD data analysis and for all other experiments the b-spectra are shown in grey.


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UV-visible spectra of cMbCO and the supernatant from a pelleted cMbCO sample. The supernatant (grey) shows no significant absorbance from metMb or MbCO, as indicated by the lines at 408 and 424 nm. In contrast, a typical TRLD cellular sample has a spectrum that is characteristic of MbCO (black), as well as substantial scattering. The inset shows that the Mb sample before CO is introduced comprises a mixture of metMb and oxygen-bound Mb, resulting in an absorbance maximum of 413 nm. 84x64mm (300 x 300 DPI)


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Microviscosity in E. coli Cells from Time-Resolved Linear Dichroism

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