Single-Molecule FRET Measurements in Additive-Enriched Aqueous

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Single-molecule FRET measurements in additive-enriched aqueous solutions Daryan Kempe, Michele Cerminara, Simón Poblete, Antonie Schöne, Matteo Gabba, and Joerg Fitter Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03147 • Publication Date (Web): 29 Nov 2016 Downloaded from http://pubs.acs.org on November 29, 2016

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Analytical Chemistry

Single-molecule FRET measurements in additiveenriched aqueous solutions Daryan Kempe†*, Michele Cerminara‡, Simón Poblete§•, Antonie Schöne‡, Matteo Gabba‡••, and Jörg Fitter†‡* †

AG Biophysik, I. Physikalisches Institut (IA), RWTH Aachen University, 52056 Aachen, Germany

‡

Molecular Biophysics, Institute of Complex Systems (ICS-5), Forschungszentrum Jülich, 52428 Jülich, Germany

§

Theoretical Soft Matter and Biophysics, Institute of Complex Systems (ICS-2), Forschungszentrum Jülich, 52428 Jülich, Germany *[email protected] *[email protected] Fax: + 49 241 80 22331 ABSTRACT: The addition of high amounts of chemical denaturants, salts, viscosity enhancers or macro-molecular crowding agents has an impact on the physical properties of buffer solutions. Among others, the (microscopic) viscosity, the refractive index, the dielectric constant and the ionic strength can be affected. Here, we systematically evaluate the importance of solvent characteristics with respect to single-molecule FRET (smFRET) data. First, we present a confocal based method for the determination of fluorescence quantum yields to facilitate a fast characterization of smFRETsamples at sub-nM-concentrations. As a case study, we analyze smFRET data of structurally rigid, double-stranded DNAoligonucleotides in aqueous buffer and in buffers with specific amounts of glycerol, guanidine hydrochloride (GdnHCl) and sodium chloride (NaCl) added. We show that the calculation of inter-dye distances, without taking into account solvent-induced spectral and photo-physical changes of the labels, leads to deviations of up to 4 Å from the real inter-dye distances. Additionally, we demonstrate that electrostatic dye-dye repulsions are negligible for the inter-dye distance regime considered here (>50 Å). Finally, we use our approach to validate the further compaction of the already unfolded state of Phosphoglycerate Kinase (PGK) with decreasing denaturant concentrations, a mechanism known as coil-globule transition.

with the characteristics of the surrounding environment, for instance with the refractive index, the viscosity and the polarity.17-20 Many biophysical experiments result in drastic changes of those properties. For example, considering protein (un)folding experiments, concentrations of up to 6 M of the chemical denaturant GdnHCl are frequently employed, changing the refractive index n from 1.33 to 1.43.21 Simultaneously, the dielectric constant decreases by more than 50 % whereas the viscosity increases by a factor of 1.6 as compared to aqueous buffer.22,23 Furthermore, the determination of thermodynamic parameters describing the salt dependent duplex formation of DNA, or of the effect of Hofmeister ions on alpha-helical protein structures, is coupled to the use of saltconcentrations in the molar range,24,25 which implies a decrease of the Debye length and of the dielectric constant.26 In addition, to stabilize samples or elongate the observation time window of smFRET measurements, specific amounts of Glycerol can be added to

Introduction: Over the last few decades, single-molecule FRET has become a valuable tool enlightening the fields of molecular conformational dynamics, folding and structure determination.1-5 As FRET is based on the dipole-dipole coupling of the transition dipole moments of a donor and an acceptor fluorophore, the extent of energy transfer is strongly distance dependent.6 Therefore, attaching donor and acceptor to specific positions of a (biological) specimen, FRET can be used as a “spectroscopic ruler”, monitoring inter-dye distances in the nm range.7-11 In addition, by analysis of the variance of the obtained FRET efficiency histograms, information about the underlying molecular dynamics can be deduced.12-15 Quantitative statements about any parameter obtained using FRET are based on a precise calibration of the acquired data, among others taking into account the properties of the FRET-pair used.16 Its spectral, photo-physical as well as electrostatic signature can vary significantly 1

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solutions,27,28 which has a strong effect on the refractive index and viscosity of the solution.29 Particularly in the recent past, buffers crowded with macro-molecular agents like Polyethylenglycol (PEG), Dextran or Ficoll have been frequently used to artificially mimic the cytosol of the cell,30,31 resulting in changes of the refractive index and of the microscopic viscosity that can be hard to assess.32 Going one step further, even the combined effect of variations in crowding agents and solution conditions on intrinsically disordered proteins was studied by smFRET just recently.33 Obviously, a large number of experiments are commonly done in additive-enriched aqueous solutions, making the evaluation of possible solvent-induced artefacts in smFRET data an issue of relevance. Furthermore, several comparative studies of GdnHCl induced unfolded states of proteins showed a significant disagreement between smFRET and small-angle X-ray scattering (SAXS) data. This raises the question whether the compaction of the unfolded state upon decrease of denaturant concentration (coil-globule transition) observed by smFRET might be a consequence of disregarded calibrations or electrostatic repulsions between the negatively charged fluorescent markers employed in all cases.34-36 In the present work, we systematically evaluate the importance of solvent characteristics with respect to smFRET parameters. We use double stranded DNA oligonucleotides to assess the importance of electrostatic dye-dye interactions in a distance regime typically considered in protein unfolding measurements. We complement our experimentally obtained inter-dye distances with theoretical predictions taking into account electrostatic dye-dye repulsions. Furthermore, aiming to rule out that the discrepancy between scattering and smFRET measurements of denatured proteins originates from artifacts due to the complex composition of the solution, we monitor the impact of denaturant on the two-domain protein Phosphoglycerate Kinase (PGK). By eliminating possible artifacts in the inter-dye distance determination, we validate the existence of a coil-globule transition beyond the classical unfolding transition.

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purchased from Purimex (Grebenstein, Germany). Unlabeled single strands were purchased from Eurofins (Aachen, Germany). Details on sequences are given in the SI. All measurements of DNA oligonucleotides were performed in DNA-buffer (20 mM TRIS, 100 mM NaCl, 10 mM MgCl2, pH = 7.5) with or without the respective additives. Phosphoglycerate Kinase (PGK) from Saccharomyces cerevisiae with mutations S1C and Q135C was prepared and labeled with AL488 and AL647 as described in ref.39. Additionally, the corresponding single-cysteine mutants were prepared and labeled for control measurements. PGK-buffer contained 50 mM MOPS, 50 mM NaCl and 0.005 % Tween20 (Sigma-Aldrich, St. Louis, USA) at a pH = 7.4. For the unfolding experiments, specific amounts of GdnHCl were added. The concentrations of denaturant solutions were adjusted by refractive index measurements.21 The viscosity of each solution was determined according to ref.23. Additionally, a photo-protection cocktail (1 mM Trolox, 10 mM Cysteamine) was added to all solutions. Instrumentation: The refractive indices of all solutions were determined using an Abbe refractometer (A. Krüss Optronic, Hamburg, Germany). Absorption spectra were recorded using a Shimadzu (Kyoto, Japan) UV-2600 double-beam UV-VIS spectrophotometer. Fluorescence spectra were determined with a PTI (Bensheim, Germany) QuantaMaster40 spectrofluorometer. CD spectra in the far UV-region (200-280 nm) were acquired with a JASCO (Gross-Umstadt, Germany) J-1100 spectropolarimeter. Fluorescence Correlation Spectroscopy (FCS) and smFRET measurements were performed with a commercial confocal microscope (MicroTime200, Picoquant, Berlin, Germany) equipped with a red (640nm) and a blue (485nm) diode laser and an UPLSAPO 60x/1.2NA objective from Olympus (Shinjuku, Japan). Details on the apparatus are given in the SI. FCS curves were generated and analyzed with the help of the software Symphotime64 from PicoQuant. Time-resolved anisotropy (TRA) and FRET datasets were analyzed with self-written Matlab (Mathworks, Nattick, USA) routines. All other data-sets were processed with Origin Pro (Northampton, USA). Further details on the data analysis are given in the referring subsection of the SI.

Experimental Section: Materials: Alexa Fluor 488-C5 maleimide/NHS-Ester (AL488) and Alexa Fluor 647-C2 maleimide/ NHS Ester (AL647) were purchased from ThermoFisher Scientific (Waltham, USA). Atto488-NHS-Ester (AT488) and Atto655-NHS-Ester (AT655) were purchased from AttoTec (Siegen, Germany). Glycerol solutions were prepared by adding the specific weight fraction of Glycerol (SigmaAldrich, St. Louis, USA) to ultrapure water or buffer. The solutions were sealed and stirred at 400 rpm for 30 minutes at room temperature. The viscosities of the glycerol solutions were estimated according to their concentrations.29 Concentrations were additionally validated by refractive index measurements.37 Double-stranded DNA (dsDNA) samples were prepared by hybridization of complementary single-strands as previously described.38 Single-strands either labeled with AL488 or AL647 were

Methods: Quantum yield determination: The conceptual basis of the fluorescence quantum yield (QY) determination employing FCS in the limit of low excitation intensities is described in detail in ref.40,41 To extend this method to the case of additive-enriched solutions, some crucial precautions need to be taken, as described in detail in the SI. Weighted accessible volume calculations taking into account electrostatic dye-dye interactions: Accessible volume (AV) calculations are a common approach to predict the sterically accessible space of a fluorescent marker attached to a macro-molecule.10 Being a 2


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purely geometrical search algorithm, no physical dye-dye or dye-macro-molecule interactions are taken into account. For this work, we modified our AV calculation routine to consider not only steric clashes but also the effect of the total charge of the dyes, including the screening of their mutual repulsion due to the solvent environment, as explained in detail in the SI.

denotes the absorption coefficient at the excitation laser wavelength and refers to the slope of the molecular brightness curve.40 The subscripts denote the reference sample, R, and the sample of interest, S. stands for the integrated molecule detection function of the setup and can, as distinct from the pure geometry of the MDF, be affected by a refractive index mismatch. This influence can be quantified by rearranging eq (1):

Results and Discussion:

∙ ∙ ∙ 2

Confocal quantum yield determination in additiveenriched solutions: The quantitative analysis of FCS curves relies on the precise knowledge of the geometrical parameters describing the molecule detection function (MDF) of the confocal setup. Here, we determined the effective volume V and the ellipticity in dependency of an increasing refractive index using glycerol solutions of AL488 with known diffusion coefficient D (details on the determination of D are given in the SI)42. The results are depicted in Figure 1, showing that both geometrical parameters describing the MDF stay, within the given error limits, constant for the refractive index regime considered here. Hence, under the applied experimental conditions of a short coverslip-focus distance and a small pinhole diameter, the refractive index mismatch has a minor effect on the Gaussian shape of the MDF. Consequently, the general accuracy of the FCS analysis and the related confocal QY determination are not impaired. On top of that, the determination of D for specimen in solutions of high refractive index requires only one MDF calibration measurement in a solvent of arbitrary refractive index.

and determining the ratio of the s using a fluorophore (AL488, AL647) diffusing in water as R, and the same fluorophore in glycerol as S. All parameters entering eq (2) were determined for all solutions. QYs were measured with an approach based on steady-state absorption and fluorescence spectroscopy,43 further on called the standard method (see Figure 2 (a)). While the QY of AL488 stays, within the given error limits, constant for all glycerol solutions, the values for AL647 significantly increase with the refractive index. This is most likely a result of the increasing viscosity of the glycerol solutions, reducing the cis/trans transition rate of AL647.44 The corresponding ratios are depicted in Figure 2 (b). Independent of the spectral range, we see a significant ratio with increasing refractive indidecrease of the ces. Therefore, neglecting its contribution would lead to a remarkable error up to 40 % in the QY determination. Consequently, it is absolutely necessary to measure reference and sample in solutions of same refractive index to determine accurate fluorescence QYs.

Figure 1: Ellipticity and volume of the molecule detection function as a function of refractive index n: AL488 measured in glycerol solutions (10µm coverslip-focus distance, 30µm pinhole). Within the given error limits, both parameters stay constant for the regime considered here.

Figure 2: (a) The fluorescence QY of AL488 dissolved in glycerol solutions is independent from the refractive index. As distinct from that, the fluorescence QY of AL647 significantly increases with increasing amounts of glycerol added to the solution. (b) The ratio of the s measured in water and in solutions of increasing refractive indices for the blue and red spectral regime clearly changes with n. Solid lines depict spline interpolated values. The experimental values are reported in Table S-2.

The fundamental equation needed for the confocal QY determination is given by:

∙ ∙ ∙ ∙ 1

Here, equals the transmission efficiency of the setup,

To finally validate our approach, we determined the QYs of AT488 and AT655 in glycerol solutions of refractive 3



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indices n=1.34 and n=1.37. AL488 and AL647 dissolved in glycerol solutions of same refractive indices were used as reference samples. Within the error limits, the results perfectly coincided with the standard method (see Figure 3), supporting the excellent reliability of the confocal detection method.

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protein) in the same solvent. However, even in this case it is not sure that the natural lifetime of the fluorescent marker is not influenced by its local interaction with the surface of the target molecule. Additionally, static quenching is never visible using the lifetime method, making a complementary way of QY determination inevitable. FRET studies on DNA-oligonucleotides in additive-enriched environments: In this section we want to quantify the impact of the solvent characteristics on the inter-dye distances determined by smFRET. As test specimen, we used structurally rigid, dsDNA oligonucleotides with donor and acceptor linkers attached at a distance of 10bp and 17bp. In Figure 5 (a), the 3D structure of the constructs and the corresponding AVs of the donor and acceptor fluorophore are shown.10,46 As solvents we used DNA buffer and DNA buffer with either 1 M NaCl, 4 M GdnHCl, or 50 wt. % Glycerol added. Fluorescence emis-

Figure 3: QY-values of AT488 (left) and AT655 (right) obtained with our confocal and the standard method are in perfect agreement.

sion and absorption spectra of all samples showed a pronounced red-shift going from pure buffer to GdnHCl and Glycerol, but did not change upon the addition of 1M NaCl (Figure S2). The fluorescence lifetime of the donor attached to DNA decreased going from buffer to Glycerol or denaturant solution, whereas the lifetime of the acceptor increased (Figure S3). The quantum yields of donor and acceptor were determined as described above (results in Table S-4). FRET efficiency histograms were calculated according to:16

& %

3 & &

& ' ∙ ∙ ( ( (

At last, we compared our approach to the commonly used determination of QYs based on the fluorescence lifetime, (details in the SI). To test the performance of the lifetime method in solutions of high refractive indices, we determined the ratio of the natural lifetimes, , / , , for AL488 and AL647 in glycerol solutions, using the same fluorophores dissolved in water as reference samples.

Here, &,( denotes the numbers of detected acceptor, donor photons per selected burst (find details on burst selection in the SI). In Figure 5 (b), the FRET efficiency histograms taking into account all solvent induced spectral and QY changes (corrected) are compared to the ones calculated with the parameters measured in DNA buffer (uncorrected). All histograms were fitted with a Gaussian distribution to determine the mean FRET efficiency 〈%〉. The deviation between corrected and uncorrected 〈%〉values spans from 10 % to remarkable 35 % for 4 M GdnHCl (results in Table S-4). Additionally, almost all uncorrected histograms show a misleading apparent increase of the transfer efficiency. Subsequently, to show the combined effect of Förster radius , and efficiency corrections, the mean inter-dye distances in all solvents were calculated. To calculate the Förster radius, we need to assess the value of the orientational factor, # . Therefore, we used time-resolved anisotropy (TRA) to determine the rotational mobility of the linker-dye unit. All obtained parameters are given in Table S-5 along with a detailed discussion about the corresponding # values. In the following, all inter-dye distances were calculated us-

Figure 4: Ratio of the natural lifetimes of reference and sample for AL488 and AL647 plotted as a function of the ratio of the refractive indices squared. Whereas AL488 shows a linear dependency according to the rule of Strickler-Berg, AL647 shows a close to parabolic behavior.

The obtained results are depicted in Figure 4. Plotting , / , as a function of " # /"# , we demonstrate that the ratio for AL488 approximates the linear prediction of Strickler-Berg.45 On the contrary, for AL647 a close to parabolic shape is found. Obviously, the QY determina-

tion using the fluorophores in water as reference is not possible, as it is not known a priori how to correct the influence of the solvent on the natural lifetimes. To circumvent this problem, one could try to measure reference (free dye) and sample of interest (e.g. labeled 4


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ing =2/3 (Förster radii in Table S-6). It is worth men-

tioning that, with the exception

Figure 5: (a) 3D structures of dsDNA oligonucleotides; 10bp and 17bp are the distances in between linker attachment points. In red, the sterically accessible volume (AV) of the acceptor is shown. The AV of the donor is depicted in blue. (b) FRET-efficiency histograms of the two constructs in different solvents. The corrected histograms are calculated taking into account spectral and QY changes of the FRET pair, whereas the uncorrected histograms are calculated with the parameters determined for buffer. Black solid lines depict the Gaussian distributions best fitting the data. Deviations of up to 35 % are found along with a misleading apparent increase of 〈-〉 for the uncorrected data. The 3D structures of the constructs were generated using 3D-DART.

of the donor in Glycerol, the relevant anisotropy parameters do not change significantly going from the 10bp to the 17bp construct. Consequently, should be similar in both cases, enabling us to monitor relative distance changes correctly even if we estimated erroneously. In Figure 6, we report the inter-dye distances ,(& for the two dsDNA constructs calculated in three different ways: (i) the fully corrected ,(& for different solvent compositions, taking into account all solvent-induced changes; (ii) ,(& calculated using the uncorrected FRETefficiencies as well as the Förster radius measured in buffer; (iii) ,(& calculated from the uncorrected transfer efficiencies and using the Förster radius obtained with the overlap integral measured in buffer but taking into account the actual refractive index of the solvent. The mean inter-dye distances according to AV calculations, which coincide with our experimental results obtained for dsDNA in pure buffer, are also shown. As compared to pure

aqueous buffer, the corrected values for all other environmental conditions exhibit marginally larger inter-dye distances. However, especially for the solvents containing large amounts of additives, we do observe significant differences between corrected and uncorrected ,(& -values as high as 4 Å. Additionally, the uncorrected inter-dye distances lead to a misinterpretation of the experimental results, as the oligonucleotide constructs appear to become more compact instead of more expanded in the GdnHCl and Glycerol solutions. Interestingly, including the refractive index n of the solvent and concurrently neglecting any further corrections can lead to even stronger deviations from the fully corrected inter-dye distances than applying no corrections at all. Since the impact of the individual corrections can typically not be predicted a priori, the application of all corrections is mandatory in order to obtain reliable ,(& - values. 5



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assumption that there is no antagonistic concurrent conformational change of the dsDNA constructs, the impact of electrostatic interactions on the inter-dye distance is therefore small. Additionally, the inter-dye distances increase in a similar manner for the 10bp and 17bp dsDNA construct going from buffer to the additive-enriched solutions, which should also not be the case if strongly distance dependent electrostatic interactions would play a role. To assess the impact of electrostatic interactions over a broader distance interval, we calculated inter-dye distances between spherical AV clouds in the absence of dsDNA, assuming two different charge distribution scenarios (see referring Methods section in SI). The results are depicted in Figure 7 (a). As black dotted line, interdye distances calculated without any restrictions are given. Interestingly, these don’t coincide with the centroid distances (solid black line) but show a constant positive offset. This mismatch is mainly due to the geometry of the clouds (see eq (8) in the SI for details). Remarkably, it is present even in the absence of clashes and electrostatic repulsions. It does, however, vanish in the limit of large distances between the tethering points (data not shown here).

Figure 6: Mean inter-dye distances /0 of 10bp and 17bp dsDNA constructs in different solvents [c(NaCl)=0.1 M, c(GdnHCl)=4 M, 50 wt. % Glycerol]. In violet, the fully corrected distances are shown. In green, /0 calculated using uncorrected 〈-〉 and /1 are depicted. In green, distances determined using uncorrected 〈-〉 and /1 values, the latter corrected for solvent refractive indices, are shown. Error-bars are determined assuming an error of 2 5% of 340 and 34 , calculating the corresponding - distributions, fitting them with a Gaussian distribution and calculating the upper and lower limit of /0 using eq (9) in the SI. The violet dashed lines represent the error limits for the measurements in buffer. All /0 values are summarized in Table S-7.

Inter-dye distance calculations taking into account electrostatic dye-dye interactions: Going from pure aqueous buffer to the other solvent environments, the dielectric constant as well as the Debye length decrease (the physical properties of buffer solutions relevant for this work are summarized in Table S-2). In this section, we want to have a closer look at the impact of these two parameters on the resulting inter-dye distances. At first, we calculated the AVs of AL488 and AL647 attached to the dsDNA constructs (see Figure 5 (a)) and weighted each point of the AV according to its electrostatic interaction potential (for details see SI). The resulting average inter-dye distances (Table S-8) coincide for all the solvent conditions considered, hence being quite insensitive to electrostatic dye-dye repulsions. This is in accordance with the experimental findings from the smFRET measurements of the dsDNA constructs. As shown in Figure 6, for both distance regimes the increase of the inter-dye distance lies between 1 and 2 5 going from pure aqueous buffer to complex solvent conditions. Comparing the additive-enriched solutions only, the resulting inter distances even coincide within their error-limits. Under the

Figure 7: (a) Inter-dye distances /0,0 plotted as a function of the distance 67 between the centroids of the spherical AVs of donor and acceptor. As black solid line, /0,0 67 is depicted. The dotted black line shows the inter-dye distance calculated without any restrictions, whereas the dashed black line is calculated excluding steric clashes (partly hidden by solid green line). As green solid (NaCl) and dasheddotted (Glycerol) lines, the inter-dye distances corresponding to the isotropic electrostatic scenario are given, whereas the solid and dashed-dotted orange lines represent the results based on the anisotropic electrostatic scenario. (b) Directly calculated inter-dye distances (green) and FRETefficiency based inter-dye distances (magenta) plotted as a function of the distance 67 . Electrostatic interactions correspond to the isotropic regime.

Inter-dye distances calculated excluding steric clashes are very similar to the inter-dye distances obtained for NaCl and Glycerol, calculated excluding sterical clashes and taking into account electrostatic interactions correspond6


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ing to the isotropic scenario. Clearly, the effect of sterical clashes outweighs the effect of electrostatic repulsions in our approximation. Additionally, the difference between the resulting ,(&,&8 values for NaCl and Glycerol is smaller than 1 5 considering the whole distance regime. Furthermore, we tested the anisotropic scenario since it represents a kind of extreme case of electrostatic interaction and can give an idea of upper boundaries concerning inter-dye distance changes. Even here, the maximal difference between ,(&,&8 for NaCl and Glycerol lies around 3 5. As a last step, we compared the inter-dye distances directly calculated as before to the ones obtained by previous determination of FRET efficiencies and subsequent calculation of the corresponding distances (see SI for theoretical derivation). Here, we assumed an isotropic charge distribution again and compared the cases of NaCl and Glycerol (results shown in Figure 7 (b)). Obviously, the inter-dye distances taken from FRET efficiencies show a different pattern than the directly calculated ones. However, in this case we can see that the contribution of sole electrostatic interactions is even smaller than before. Finally, using these rough approximations, we can state that even for short inter-dye distances around 30 5 a change of the solvent environment leads to a relative difference in ,(& of below 10 %. Nonetheless, additional effects such as solvent mediated interactions and higher resolution electrostatic contributions could still play a significant role and should be considered separately.47 Coil-globule transition of PGK: As another commonly applied test case, we studied the denaturant induced intra-domain unfolding of a two- domain protein, i.e. PGK. As before, AL488 and AL647 were chosen as donor and acceptor fluorophores and attached to position 1 and 135 of the N-terminal domain (Figure 8 (a)). In addition to pure aqueous buffer, we employed buffer solutions with high amounts of GdnHCl added (1 M, 1.5 M, 4 M and 6 M). Going from low to high concentrations of denaturant, steady-state absorption and fluorescence emission spectra of donor and acceptor showed a pronounced red shift (Figure S7). In the case of the acceptor, the fluorescence lifetime increased with increasing amounts of GdnHCl added, whereas the lifetime of the donor changed rather insignificantly (Figure S8). As before, to be able to calculate FRET efficiency histograms, we determined the QYs and spectra of donor and acceptor attached to the protein in all solvents. The obtained ( and & are similar to the values obtained for dsDNA in 4 M GdnHCl, indicating that rather the solvent properties than the local protein environment determines QY variations (Table S-9). The corresponding corrected and uncorrected FRET efficiency histograms are shown in Figure 8 (b). Strikingly, in all cases, the mean FRET-efficiencies of corrected and uncorrected histograms differ by more than 30 % (Table S-9). Furthermore, the widths of the histograms show relevant discrepancies.

Figure 8: (a) Crystal structure of PGK [PDB:1QPG] superimposed with the AVs of AL488 and AL647. Attachment positions at S1C and Q135C are shown as dark spheres. (b) FRETefficiency histograms of PGK in buffer with increasing amounts of GdnHCl added. The corrected histograms are calculated taking into account spectral and QY changes of the FRET pair, whereas the uncorrected histograms are calculated with the parameters determined for pure buffer. Black solid lines denote the Gaussian distributions best fitting the data. Black dashed lines depict the shot-noise lim48 ited distributions. The vertical lines indicate the mean FRET-efficiency of the already unfolded state at 1M GdnHCl.time-scale of FRET in all cases.

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tances, and n corrected distances. Here, only at 1.5 M GdnHCl significant differences between corrected and uncorrected ,(& - values show up (see also Table S-12). Again, correcting for n only is not advisable. Although the protein is almost completely unfolded at1 M GdnHCl with respect to secondary structure elements (see Figure S11), we observe an ongoing further expansion of the protein structure in the regime from 1 M to 6 M GdnHCl. This phenomenon is well pronounced in the corrected as well as in the uncorrected ,(& values and was already observed for many other proteins (coil-globule transition going from high to low denaturant concentrations).34,36,4951 In addition, (viscosity-corrected) hydrodynamic radii RH determined by FCS reflect exactly the same behavior (Figure 9(b), for details on viscosity determination see Figure S9 in the SI). Although for the PGK mutant the overall impact of the applied corrections is small as compared to the dsDNA samples, we now can be confident about the development of inter-dye distances for this protein structure under highly denaturing conditions.

Especially at 1 M GdnHCl, the width of the corrected histogram shows a 40 % smaller deviation from the width of the shot-noise limited distribution than the corresponding width of the uncorrected histogram. This is of crucial importance for wide-spread analysis tools comparing variance of the data to get insight into the underlying molecular dynamics.12,14 To assess the orientation factor # , we employed TRA measurements as before. Here, the rotational mobility of donor and acceptor appears to trace the denaturant induced structural changes of PGK: the mobility of the dyes increases upon unfolding (which is reflected in both, the correlation time theta and the semicone angle alpha, see Figure S10). For the donor, the obtained correlation times are smaller than the inverse FRET rate, whereas for the acceptor 9: is on the timescale of FRET in all cases. However, assuming # =2/3, the determined ,(& for PGK in aqueous buffer coincided with the distance predicted by AV calculations (see Figure 9 (b)). Therefore, the rather slow rotational correlation time of the acceptor does not

Conclusions: In the present study, we demonstrated that high amounts of additives can alter the properties of aqueous solutions, fluorescent markers as well as the confocal detection scheme. We presented approaches based on adjusted experimental settings and on calibration measurements which allow us to obtain reliable results from FCS and FRET experiments. With respect to FRET studies, in particular the explicit determination of correct quantum yields for all attachment positions and for all environmental conditions is essential. In particular for smFRET studies of proteins, attached dyes are not only affected by the specific solvent properties but also by potential interactions with the local polypeptide chain environment. This can lead to a drastic deviation of quantum yields compared to those typically known for the free dye in aqueous buffer solutions. Therefore, a simple interpolation between values obtained at low and at high cosolvent concentrations would be erroneous, meaning that each value at each environmental condition has to be measured explicitly. From the magnitude of the individually observed parameters (& ,( , , ) it is not straightforward to estimate the magnitude of the overall correction in terms of ,(& . Beyond that, the application of corrections for only one or a few parameters can be hazardous because it can give rise to even stronger deviations from the fully corrected ,(& -values. A special case of interest where potential artefacts and required corrections in smFRET results play an important role is given by the question which structural properties unfolded proteins develop upon increasing denaturant concentrations beyond the unfolding transition. Recently, a detailed description of the conformational ensemble of denatured ubiquitin was obtained by comparing smFRET data with ensembles computed using NMR/SAXS restrains, confirming the occurrence of the coil-globule transition.52 However, since for many single domain proteins such an

Figure 9: (a) Inter-dye distances (RDA) of PGK at high GdnHCl concentrations: fully corrected (violet), uncorrected (orange) and corrected n only (green) (b) fully corrected RDA values compared to hydrodynamic radii RH from FCS data. The unfolding transition is indicated by a two-state fit with a mid-point at ~0.7 M GdnHCl (dashed-lines), while the coilglobule transition is illustrated by the straight dotted lines. As red line, the inter-dye distance obtained by AV calculations is shown.

seem to interfere with the assumption of dynamic orientational averaging. Consequently, we determined all distances using # =2/3 (Förster radii in Table S-11). The obtained results are shown in Figure 9 (a). As before, we determined fully corrected distances, uncorrected dis8


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Analytical Chemistry solutions; Quantum yields and FRET efficiencies of dsDNA constructs; Rotational correlation times and semi-cone angles of dsDNA constructs; Förster Radii of dsDNA constructs; Inter-dye distances of dsDNA constructs; Inter-dye distances of dsDNA constructs determined by AVcalculations considering electrostatic interactions; Quantum yields and FRET efficiencies of PGK; Rotational correlation times and semi-cone angles of PGK; Förster Radii of PGK; Inter-dye distances of PGK

ongoing expansion with increasing denaturant concentrations is not observed by small-angle X-ray scattering (see 34 and references therein), the question arises whether in smFRET studies methodical deficiencies systematically bias the obtained results. From our analysis on a mutant of y-PGK we observe a clear coil-globule transition behavior at elevated GdnHCl concentrations. This result gives, on the one hand, additional reliability qualified by the application of a thorough parameter correction and, on the other hand, by an independent measure of the hydrodynamic radii under identical sample conditions. Possible inter-dye interactions caused by electrostatics between charged dyes (both employed dyes carry net-charges) and modified by different types of solvent (with different dielectric permittivity and ionic strength) were shown to have no impact on our results. In reference measurements of dsDNA constructs with inter-dye distances of 49 Å and 61 Å, we observed only small differences in interdye distances between pure buffer and buffer of high GdnHCl concentration. Consequently, electrostatic dyedye repulsions do not explain the GdnHCl induced increase of inter-dye distances we observe for PGK. In contrast to the PGK mutant considered here, most of the other proteins for which a pronounced coil-globule transition was observed were much smaller and therefore, the native inter-dye distance was also smaller (i.e. well below the Förster radius of~ 50 Å). However, for the unfolded states, the observed inter-dye distances with transfer efficiency values between 0.4 -0.7 fell into the same distance regime as considered in the example of dsDNA. Consequently, also in these cases we can be confident that electrostatic dye-dye interactions are negligible. Finally, we can state that the presented methodical approach is not only applicable for the rather simple buffer solutions which we investigated in this study, but also for more complex buffer compositions consisting of macromolecular crowding agents or even cytosolic buffers.

This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors: *[email protected] *[email protected]

Fax: + 49 241 80 22331; Tel: +49 241 80 20299 Present Addresses •

Molecular and Statistical Biophysics, Scuola Internationale Superiore di Studi Avanzati (SISSA), 34136 Trieste, Italy

••

Membrane Enzymology Group, Groningen Institute of Biomolecular Sciences & Biotechnology, Faculty of Mathematics and Natural Sciences, 9747 AG Groningen, The Netherlands

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors would like to especially thank Dr. Tina Züchner for the critical revision of the manuscript. The authors would also like to thank Henning Höfig for his advice regarding the AV calculations and Friedemann Landmesser for his support in the performance of TRA and FCS measurements of AL488 in Glycerol. Special thanks go to Ilona Ritter and Julia Walter for competent technical support. A. S. thanks the International Helmholtz Research School on Biophysics and Soft Matter (BioSoft) for financial support.

ASSOCIATED CONTENT Supporting Information. Materials: Oligonucleotide sequences Instrumentation: Confocal microscope setup Methods: Confocal quantum yield determination, Lifetimebased quantum yield determination, Accessible volume calculations taking into account electrostatic interactions Data analysis: Fluorescence correlation spectroscopy (FCS), Time-resolved anisotropy (TRA), Single-molecule FRET Figures: Quantum yield determination, steady-state and confocal; Spectra DNA; Lifetimes DNA; Dye charge distribution models; Rotational correlation times of AL488 in glycerol solutions; Exemplary TRA decay of dsDNA-AL488 in 4M GdnHCl; Spectra PGK; Lifetimes PGK; Dynamic viscosity of GdnHCl solutions; Rotational correlation times and semicone angles of AL488 and AL647 attached to PGK in GdnHCl-solutions; Unfolding of PGK monitored by circular dichroism (CD) spectroscopy Tables: Dye-linker parameterization; Physical properties of solvents used; Refractive indices and viscosities of glycerol

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Single-Molecule FRET Measurements in Additive-Enriched Aqueous

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