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Highly Heterogeneous Nature of the Native and Unfolded States of B Domain of Protein A Revealed by TwoDimensional Fluorescence Lifetime Correlation Spectroscopy Takuhiro Otosu, Kunihiko Ishii, Hiroyuki Oikawa, Munehito Arai, Satoshi Takahashi, and Tahei Tahara J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 10 May 2017 Downloaded from http://pubs.acs.org on May 15, 2017

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The Journal of Physical Chemistry

Highly Heterogeneous Nature of the Native and Unfolded States of B Domain of Protein A Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy

Takuhiro Otosu†, ∇, Kunihiko Ishii†, §, Hiroyuki Oikawa⊥, Munehito Arai||, Satoshi Takahashi⊥, and Tahei Tahara*,†, §

†

Molecular Spectroscopy Laboratory, RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198,

Japan §

Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics (RAP),

RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan ⊥

Institute of Multidisciplinary Research for Advanced Materials, Tohoku University,

2-1-1 Katahira, Aoba, Sendai, Miyagi 980-8577, Japan ||

Department of Life Sciences, Graduate School of Arts and Sciences, The University of

Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan

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ACS Paragon Plus Environment


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ABSTRACT

Elucidating the protein folding mechanism is crucial to understand how proteins acquire their unique structures to realize various biological functions. With this aim, the folding/unfolding of small globular proteins has been extensively studied. Interestingly, recent studies have revealed that even such small proteins represent considerably complex processes. In this study, we examined the folding/unfolding process of a small α-helical protein, the B domain of protein A (BdpA), in an equilibrium condition using two-dimensional fluorescence lifetime correlation spectroscopy with 10-microsecond time resolution. The results showed that, although the BdpA is a two-state folder, both the native and unfolded states are highly heterogeneous and the conformational conversion within each ensemble occurs within 10 microseconds. Furthermore, it was indicated that the average structures of both ensembles gradually change and get more elongated as the denaturant concentration increases. The analysis on two FRET mutants suggested that fraying of N-terminal helix is the origin of the inhomogeneity of the native state. Because the direct observation of the ensemble nature of the native state at the single molecule level has not been reported, the data obtained in this study give new insights into complex conformational properties of small proteins.

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INTRODUCTION

Proteins are chains of amino acids which spontaneously fold into specific structures to acquire various biological functions. Evolution selects such unique structures as the most stable and minimally frustrated states under physiological conditions.1 If misfolding of proteins occurs in our body, it may cause serious diseases.2-4 Understanding the folding mechanism of proteins is thus crucial to elucidate how proteins fold into the correct structure and under what conditions misfolding may occur. The simplest model of folding is the two-state model in which a protein folds cooperatively by crossing a single free-energy barrier between the native and unfolded states.5-8 Indeed, extensive studies have shown that a small, single-domain globular protein (which consists of less than 100 amino acids) usually folds/unfolds in a two-state manner without appearance of any distinct intermediate states.7, 9-12 Recently, the folding process deviated from the simple two-state model has been discussed for small proteins. For example, the downhill folding model, in which an unfolded protein folds without any barrier-crossing processes, has been proposed for a certain class of small proteins.13-15 Even for apparent two-state folders, a closer look sometimes finds gradual disordering of the native state.16-18 Theoretical-empirical

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studies also showed that for some small proteins, the folding free energy barrier is marginal and the folding process is highly sensitive to the experimental condition.19 Nevertheless, for fully elucidating such complex folding processes beyond the two-state model, a suitable experimental method is required which has both a high time-resolution (for observing fast folding processes of small proteins which sometimes occur in microseconds) and the structure resolution (for distinguishing the native, unfolded, and intermediate states). We recently developed two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), which enables us to analyze the spontaneous conformational dynamics of proteins with a microsecond time-resolution utilizing the FRET technique.20-23 In 2D FLCS, a fluorophore is attached to the target protein, and correlation of the fluorescence lifetime of the single molecule is analyzed to reveal the conformational heterogeneity of proteins as well as the interconversion dynamics among different conformers. The usefulness of 2D FLCS for protein folding studies was demonstrated by a recent work on the conformational dynamics of cytochrome c in an acidic pH condition.23 The high time-resolution and the conformational sensitivity of 2D FLCS allowed us to clarify the conformational heterogeneity of the protein and to detect microsecond conformational interconversion between the native state and an

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intermediate state. In this work, we applied 2D FLCS for investigating the folding/unfolding process and the relevant conformational properties of the B domain of protein A (BdpA). BdpA is a 60 amino-acid, α-helical protein which consists of three helices, that is, helix 1 (residues 10-19), helix 2 (residues 25-37), and helix 3 (residues 42-56) (Fig.1). BdpA has been considered a simple two-state folder.24-27 However, the folding mechanism as well as the transition-state structure of this protein remains controversial.28-37 For instance, Oikawa et al. recently performed a single-molecule line-confocal tracking experiment of FRET-labeled BdpA,28-30 and suggested that the conformation of the native state as well as that of the unfolded state is highly sensitive to the denaturant concentrations. However, this observation of the fragile conformation of the native state contradicts the conclusion of previous smFRET experiment on BdpA.31 Furthermore, theoretical investigations have failed to reproduce the transition-state structure which was determined experimentally by extensive Φ-value analyses.32-37 Thus, the folding process of BdpA has not yet been fully understood. In this study, we performed 2D FLCS on the two FRET mutants of BdpA that Oikawa et al. used. In 2D FLCS, the fluorescence lifetime of the FRET donor is examined and used as the indicator of the FRET efficiency in place of the

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acceptor/donor fluorescence intensity ratio. This lifetime-based FRET analysis provides structural information that is complementary to that obtained by the intensity-based FRET analysis.17,18,26 Actually, 2D FLCS is a new powerful method to elucidate the complex conformational properties of proteins and is expected to resolve the above-mentioned controversy on BdpA by making use of its high time-resolution and structural sensitivity. The obtained results showed that, although BdpA follows the two-state folding mechanism, both the native and unfolded states are highly heterogeneous. Furthermore, 2D FLCS showed that the structural interconversions within the native- and unfolded-state ensembles occur on the time scale shorter than 10 microseconds and that the averaged structures of both ensembles become elongated with increase of the denaturant concentration. Based on the results obtained from the two FRET mutants, we discuss the structural origin of the heterogeneity of the native-state ensemble.

EXPERIMENTAL PROCEDURES

Sample Preparation.

Two mutants (sample 5-55: K5C/Y15F/A55C, sample

22-55: Y15F/N22C/A55C) of BdpA were overexpressed by E. coli.28,29 (sample 5-55 6


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and sample 22-55 correspond to sample 2 and sample 1 in ref.29, respectively.) Y15F mutation was introduced for comparing the results with the previous fluorescence lifetime investigation, which used Y15F/N22C/A55W mutant.26 Purification of mutated BdpA and the labeling of FRET dyes on the cysteine residues are also described in refs.28 and 29. FRET dyes used for these samples are as follows: sample 5-55; Alexa488 (donor) and ATTO633 (acceptor), sample 22-55; Alexa488 (donor) and Alexa633 (acceptor). The donor and acceptor dyes were labeled to the two cysteine residues

in

an

interchangeable

way

for

both

samples,

e.g.

Cys5-Alexa488-Cys55-ATTO633 and Cys5-ATTO633-Cys55-Alexa488 coexist in the case of sample 5-55. Guanidium chloride (GdmCl) was purchased from Sigma-Aldrich. Other reagents are of analytical grade and were used without any purification. Steady-State Fluorescence Measurements.

Fluorescence spectra of the FRET

mutants of BdpA were measured with a commercial spectrofluorometer (HORIBA Fluorolog-3). Excitation wavelength was set at 480 nm (sample 5-55) or 488 nm (sample 22-55), and the fluorescence emitted from 500 nm to 700 nm was recorded at every 0.5 nm. Samples were prepared by diluting a concentrated BdpA solution with a buffer (20 mM Tris-HCl, pH 7.4) containing various concentrations of GdmCl. The final concentrations of the samples were 400 nM (sample 5-55) or 1 µM (sample 22-55).

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Fluorescence Decay Measurements and Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy (2D FLCS). Fluorescence decay measurements and 2D FLCS were performed with a home-made fluorescence correlation spectrometer.38 Briefly, the second harmonic (483 nm) generated from 966 nm output of a Ti;sapphire oscillator (Coherent Mira 900-F) was used as the excitation pulse. The excitation power was adjusted to 20 – 40 µW at the entrance of the microscope. Lower excitation power was used for sample 22-55 to avoid acceptor photobleaching. The fluorescence signals from the sample were separated from the excitation path by a dichroic mirror and passed through a confocal pinhole. Then, only the donor fluorescence was selected with an optical filter (Semrock FF01-525/45), and it was split into two by a nonpolarizing beam splitter and finally detected by two single-photon avalanche photodiodes (id Quantique id 100-20). The full-width at half-maximum of the instrumental response was ~ 50 ps. For each detected photon, macrotime, microtime and the detector number (#1 or #2) were recorded by a TCSPC module (Becker & Hickl SPC 140) and stored as the photon data. Macrotime (T) is the detection time of a photon from the start of the experiment whereas microtime (t) is the time interval between a detected photon and the corresponding excitation pulse (also referred to as an emission delay time). Only photon pairs detected by different detectors were analyzed to avoid

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the afterpulsing effect of the avalanche photodiode. Silicone spacer sandwiched with two glass coverslips was used as a sample cell. The cell was incubated with bovine serum albumin (BSA) solution in water (~ 75 µM) for more than one hour to avoid the adsorption of the sample on the glass surface. The BSA solution was replaced with the sample solution just before measurements. For the sample preparations, a concentrated BdpA solution was diluted with a buffer (20 mM Tris-HCl, pH 7.4) containing various concentrations of GdmCl. For fluorescence decay measurements, the sample concentration was set at 430 nM for sample 5-55 or at 5 µM for sample 22-55. Fluorescence decay curves were obtained by building the microtime histogram. In 2D FLCS, the concentrations of the samples were set at ~ 10 nM for both samples. A typical exposure time for collecting the photon data for 2D FLCS was 30 hours. Data Analysis.

Detailed description of 2D FLCS is given in the Supporting

Information as well as in refs. 21 and 22. Briefly, a 2D emission-delay (microtime) correlation map for a certain macrotime delay ∆T ( M ( ∆ T ; t ' , t " ) ) was constructed from the photon data by referring to the macrotime and the microtime information of each photon. In a 2D emission-delay correlation map, the horizontal axis corresponds to the microtime of the 1st photons whereas the vertical axis corresponds to that of the 2nd

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photons detected at the macrotime delay of ∆T after the 1st photons. Because M ( ∆T ; t ' , t " )

also contains the signals from uncorrelated background, the map of the

uncorrelated background was subtracted to obtain only the 2D emission-delay map for correlated signals ( M cor (∆T ; t ' , t" ) ). We used the 2D emission-delay correlation map obtained for a very large ∆T (1.0 ~ 1.2 sec.) as a reference of the uncorrelated 2D map, because the correlation is practically lost at that ∆T (Fig. S1). Thus, the obtained M cor (∆T ; t ' , t" ) represents

the intensity correlation of two photons with a macrotime

interval of ∆T emitted at microtimes t’ and t” from single molecules. We calculated M cor (∆T ; t ' , t" ) for ∆T = 1 ~ 20 µs, 50 ~ 100 µs, and 400 ~ 600 µs, and globally analyzed them by performing 2D inverse Laplace transform. With this procedure, we obtained a 2D lifetime correlation map for each ∆T as well as the independent lifetime distributions corresponding to an individual conformational state. The number of the independent lifetime distributions indicates the number of the conformational states that can be resolved at the shortest ∆T, and hence each lifetime distribution reveals the conformational properties of each state. It is because, on the assumption of FRET, a single exponential decay is expected for the donor fluorescence emitted with one fixed donor-acceptor distance, and thus the distribution of the fluorescence lifetime corresponds to the structural distribution of each state.

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Furthermore, the peak patterns in the 2D lifetime correlation map and their evolution with ∆T represent how conformational states interconvert with each other. In the actual analysis, 2D inverse Laplace transform was carried out in the form of the global 2D-MEM analysis on the correlated part of 2D emission-delay correlation maps (see Supporting Information for details). Calculation of 2D emission-delay correlation maps was performed by a code written in C, which was used as a plug-in module of Igor Pro (Wavemetrics). Other calculations were also performed by Igor Pro. Obtained 2D lifetime correlation maps were interpolated by using a spline function for a visual purpose and are shown in this paper.

RESULTS

Figure 1 shows the tertiary structure of wild-type BdpA (PDB ID 1SS1). Two FRET mutants of BdpA were prepared in this study, and their labeling positions are shown in Fig. 1. In K5C/Y15F/A55C mutant of BdpA (denoted as sample 5-55), the FRET dyes are located near the both ends of BdpA. On the other hand, Y15F/N22C/A55C mutant of BdpA (denoted as sample 22-55) possesses one FRET dye on the loop region between helix 1 and helix 2 and the other dye on the C-terminal end of helix 3. Because 11



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of the difference in the dye positions, these mutants are expected to be sensitive to different conformational changes of BdpA. Based on the structure shown in Fig. 1, the fluorescence lifetime of the donor in the native state of each mutant can be predicted using eq 1, 

  6  r R + 0  

τ DA = τ D (1 − E ) = τ D 

r6

.

(1)

6

In eq 1, τDA and τD are the fluorescence lifetimes of donor in the presence and the absence of acceptor, and E is the FRET efficiency. R0 is the Förster distance which is 50.8 Å (sample 5-55) or 53.3 Å (sample 22-55)29, and τD (the intrinsic fluorescence lifetime of Alexa 488) is reported to be 4.1 ns by manufacturer. The donor fluorescence lifetime in the native state is then roughly estimated from the distance (r) between Cα carbons of two cysteine residues at the labelling positions. In sample 5-55 (K5C/Y15F/A55C), the expected fluorescence lifetime of donor is ~ 170 ps which is detectable with our setup. (Note that the time resolution of the TCSPC system is ~ 50 ps.) On the other hand, FRET efficiency of the native state in sample 22-55 (Y15F/N22C/A55C) is almost unity because two dyes are located very close to each other (~ 9 Å). Thus, it is expected that the decay of the donor fluorescence in the native state of sample 22-55 is not time-resolved in our experiment. Keeping these in our mind, we describe the results of sample 5-55 and sample 22-55 one by one in the following.

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Conformational Dynamics of BdpA Probed by Sample 5-55.

Figure 2A shows

the steady-state fluorescence spectra of sample 5-55 with various GdmCl conditions. The fluorescence intensity of donor is increased and that of the acceptor is decreased with increasing the GdmCl concentration. This clearly indicates that the conformation of the sample 5-55 changes from a compact form to an expanded one as the concentration of GdmCl increases. To precisely examine this conformational transition process of the sample 5-55, the fluorescence decay curve of donor was measured at various GdmCl concentrations (Fig. 2B). The data show that the fluorescence decay gradually slows down with increasing the GdmCl concentration. This result is different from what is expected for a simple two-state transition, in which common two lifetime components are always observed and only their relative amplitudes change. The observed gradual change of the fluorescence decay curve indicates that a folding/unfolding process of the sample 5-55 is more complicated than the simple two-state transition. Figure 3 shows the 2D emission-delay correlation maps, 2D lifetime correlation maps, and the independent lifetime distributions corresponding to individual conformational states in sample 5-55, which were observed in the presence of 2.0 M GdmCl. The data for ∆T = 1 ~ 20 µs, 50 ~ 100 µs and 400 ~ 600 µs were globally

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analyzed, and the results for ∆T = 1 ~ 20 µs and 400 ~ 600 µs are shown. Two independent lifetime distributions (denoted as sp1 and sp2) were required to reproduce the 2D emission-delay correlation map for ∆T = 1 ~ 20 µs (Fig. 3E). The lifetime distribution in sp1 shows multiple peaks, which suggests that sp1 consists of a heterogeneous conformational mixture (i.e. conformational ensemble) and that the conversion dynamics among different conformations in sp1 occurs within the shortest ∆T, that is, ~ 10 microseconds. This rapid conformational interconversion within sp1 is also reflected in the appearance of corresponding cross peaks in the 2D lifetime correlation maps (Fig. 3C). On the other hand, the lifetime distribution in sp2 is predominantly composed of a single peak with the lifetime value of ~ 3.5 ns, which is close to the lifetime of free donor dye. Hence, sp2 is assignable to a state which has an extended conformation (in which FRET efficiency is nearly zero). The peak patterns in the 2D lifetime correlation maps are almost identical up to ∆T = 400 ~ 600 µs, although an early rise of the cross-correlation between sp1 and sp2 is recognized in the data at ∆T = 400 ~ 600 µs (Table S1). Therefore, it is concluded that the conformational transition between sp1 and sp2 takes time longer than 500 µs. To clarify the conformational origin of sp1 and sp2, 2D FLCS was performed at various concentrations of GdmCl, and the results are shown in Fig. 4. (Full sets of 2D

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maps are shown in Figs. S2-S4.) For all the concentrations of GdmCl, the obtained data are described with two independent lifetime distributions with similar peak patterns, and almost no cross peaks between them are observed up to ∆T = 400 ~ 600 µs. (Nevertheless, the very early part of the rise of cross peaks, which represent the conformational transition between sp1 and sp2, can be recognized in 2D lifetime maps at ∆T = 400 ~ 600 µs at any GdmCl concentration.) (Table S1) Importantly, however, the relative amplitudes of sp1 and sp2 on the 2D lifetime correlation maps are changed significantly depending on the GdmCl concentrations (Table. 1). The contribution from sp2 gradually increases and becomes ~ 50% at 3.0 M GdmCl which is a condition close to the folding/unfolding transition midpoint of BdpA.29 This result strongly suggests that sp1 corresponds to the native state and sp2 to the unfolded state. Almost no cross peaks (conformational transition) between the native (sp1) and the unfolded (sp2) states on the microsecond timescale is consistent with previous ensemble-averaged and smFRET experiments.27,37 Note that, in sp2, there might be a small contribution of the molecule lacking the active acceptor (i.e. donor-only molecules; Supporting Information), which is often observed in smFRET experiments as well as 2D FLCS measurements.22,27 The broad lifetime distribution of sp1 suggests that the native-state conformation of

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BdpA is highly heterogeneous, and the conformational transition in “the native-state ensemble” takes place faster than 10 microseconds. To further examine the conformational property of the native-state ensemble, Laplace transform was performed on the lifetime distribution of sp1’s obtained for various concentrations of GdmCl. The calculated fluorescence decay curve corresponding to each sp1 is shown in Fig. 5. The obtained curves clearly show that the overall fluorescence decay gradually slows down. This indicates that the overall conformation of the native-state ensemble is gradually elongated as the GdmCl concentration increases, and it is the origin of the gradual change of the ensemble-averaged fluorescence decay curves (Fig. 2B). On the other hand, it is difficult to discuss the conformational property of the unfolded state in detail for the sample 5-55 because the FRET efficiency is quite low even at low GdmCl concentrations so that the corresponding lifetime distribution (sp2) exhibits a single peak which is almost indistinguishable from the lifetime of donor-only molecules. In fact, it may have a contribution from donor-only molecules to some extent. Conformational Dynamics of BdpA Probed by Sample 22-55.

For

investigating the conformational property of the unfolded state, we used the sample 22-55. Figure 6 shows the steady-state fluorescence spectra of sample 22-55 as well as the fluorescence decay curves of the donor (detected at 502.5-547.5 nm) obtained by

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ensemble measurements at various concentrations of GdmCl. Fluorescence decay curves of the donor in Fig. 6B are normalized to match their integrated intensity over all the delay time with the donor fluorescence intensity at the corresponding wavelength region of steady-state spectrum in Fig. 6A. At the low GdmCl concentrations, the decay data shown in Fig. 6B clearly exhibit missing amplitudes, i.e. the normalized intensity at the time origin is significantly lower than that at the highest GdmCl concentration. This missing amplitude gradually decreases with increasing the GdmCl concentration, and it becomes very small at 4.0 M GdmCl where the unfolded state of BdpA is dominant. This result supports the expectation from the structure shown in Fig. 1 that the donor fluorescence in the native state is highly quenched in sample 22-55 due to the rapid energy transfer from donor to acceptor located in the close vicinity. Furthermore, the acceptor fluorescence measured with the acceptor excitation also shows quenching at low GdmCl, which suggests that the fluorescence in the native state is also partly quenched by collision effect in a non-radiative manner (Fig. S5). These results suggest that the missing amplitudes observed in Fig. 6B stem from the high FRET efficiency as well as the collisional quenching between dyes in the native state. The highly-quenched nature of the donor fluorescence (or the presence of the ‘dark’ state) in the native state of sample 22-55 was also shown in the previous work.29 Therefore, the change in the

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fluorescence lifetime of donor in sample 22-55 is mainly caused by the conformational change of the unfolded state (as well as the intermediate states if exist). Figure 7A-D show 2D lifetime correlation maps and the independent lifetime distributions of sample 22-55 obtained at 2.0 M and 4.0 M GdmCl conditions. Only 2D maps at ∆T = 1 ~ 20 µs are shown here, but no significant change in the peak patterns are observed up to ∆T ~ 500 µs (Fig. S6 and Table S2). Two independent species are identified, and the corresponding lifetime distributions are denoted as sp1' and sp2'. We assigned sp1’ to the unfolded state and sp2’ to a donor-only molecule because of the following reasons. First, these species should not originate from the native state. As is observed in Fig. 6B, the donor fluorescence in the native state of sample 22-55 is highly quenched. Thus, the contribution of the native state to the photon data is expected to be small as compared with that of the unfolded state and a donor-only molecule. Furthermore, even though a small population of the ‘emissive’ native-state may exist (it is indeed small as observed in Fig. 6B), the peak corresponding to the native state is hardly observed in the 2D lifetime correlation maps when the ‘emissive’ native-state interconverts with the ‘dark’ native-state with the timescale faster than calculated ∆T (Supporting Information and Fig. S7). Actually, the results of sample 5-55 show that the conformational transition within the native-state ensemble is faster than 10

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microseconds. Thus, it is unlikely that the observed peaks in the 2D lifetime correlation maps stem from the native state. Second, the fluorescence lifetime of sp2’ is the same as that of free donor dye, and the relative contribution of sp2' on the 2D maps remains small even at 4.0 M GdmCl concentration (less than 10% as shown in Fig. 7D). These results strongly suggest that sp2’ is due to the donor-only molecules. Note that the fluorescence signals from donor-only molecules are often observed in the smFRET data as a peak at E = 0.27,39 Consequently, it is safe to assign sp1' to the unfolded state and sp2' to a donor-only molecule. Existence of multiple peaks in sp1’ thus indicates the unfolded state is also a mixture of different conformations, and the rapid conformational interconversion in the unfolded-state ensemble occurs within 10 microseconds. GdmCl-dependence of the conformational change of the unfolded BdpA was analyzed by performing Laplace transform on the corresponding lifetime distributions (sp1'), and the results are shown in Fig. 7E. It is clearly shown that the fluorescence decay of the unfolded-state ensemble slows down as the GdmCl concentration increases. Taken together, 2D FLCS on sample 22-55 has clarified that 1) the conformational distribution of the unfolded state is significantly heterogeneous, 2) the conformational interconversion in the unfolded-state ensemble occurs faster than 10 microseconds, and 3) the averaged structure in the unfolded-state ensemble become expanded with increase

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of the GdmCl concentration.

DISCUSSION

In this study, 2D FLCS was performed on the two FRET mutants of BdpA (sample 5-55: K5C/Y15F/A55C, sample 22-55:

Y15F/N22C/A55C) to investigate the

folding/unfolding mechanism as well as relevant conformational properties of BdpA. The results show that the states appearing in the folding/unfolding process of BdpA can be separated into two, i.e., the native and unfolded states, in agreement with previous studies.24,26,27 However, the present study newly showed that both of the native and unfolded states are highly heterogeneous (as described as 'ensemble') and that the conformational transition within each ensemble occurs on the timescale shorter than 10 microseconds. Furthermore, GdmCl-dependence of the lifetime distribution of the native- and unfolded-state ensembles shows that the average conformation of each ensemble is elongated with increasing the GdmCl concentration. Deviation of the folding mechanism from the simple two-state model is one of the current topics of the folding study on small proteins.40 It has been claimed that some fast-folding proteins (which fold within ~ µs) undergo downhill folding, in which the

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folding progresses continuously and non-cooperatively without surmounting a substantial barrier of free energy.13,14 It is reported that BdpA is a two-state folder and its folding/unfolding transition takes place at ~ 1 ms at the transition midpoint.27 It is also known that this protein is one of the fastest folders under the low denaturant condition: its folding time was reported to be ~ 10 µs in the absence of denaturant which is actually close to the folding speed limit.25,34,41 Furthermore, the statistical analysis expected that proteins consisting of ~ 60 residues like BdpA have marginal folding free energy barriers.42 Therefore, the previous studies suggest that BdpA may have an intermediate folding property between two-state and downhill.19 In addition, intra-chain interactions of α-helical proteins, such as BdpA, are more localized compared to β-sheet-rich proteins, so that the degree of cooperativity is considered to be less. These characters of conformational change of BdpA should be reflected on the conformational properties observed in the present 2D-FLCS study, even if BdpA can be considered a two-state folder. In the following, we discuss several insights we obtained for BdpA, which are beyond the simple two-state model. Although it has not been fully supported by small-angle X-ray scattering experiments,43-46 a number of smFRET experiments indicated that the conformation of the unfolded state varies with the change of denaturant concentration.16,47 This

21



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conformational change of the unfolded state has been considered due to nonspecific structural change of the protein backbone.44,48-50 Particularly for BdpA, a previous smFRET experiment reported that the conformation of the unfolded state is elongated as the GdmCl concentration increases,31 as observed in the present 2D-FLCS study. In addition to this gradual structural change, the present study has also revealed the ensemble nature of the unfolded state as well as very fast conformational interconversion (< 10 µs) in the unfolded-state ensemble. It is noted that nanosecond dynamics of the unfolded states has been indicated for several proteins by both of the ensemble-averaged and single-molecule experiments on the nanosecond time scale.48,50,51 Because such nanosecond dynamics has also been observed in the intrinsically disorder proteins50 where strong specific interaction is not expected for the backbone, it is highly likely that the ensemble nature of the unfolded state of BdpA primarily originates from the nonspecific diffusive motion of the protein backbone. Nevertheless, it would still be possible that specific intermediates appear during the interconversion

process

in

the

unfolded-state

ensemble.

In fact, a

recent

temperature-jump infrared study on the isotope-labeled BdpA revealed nanosecond dynamics between the unfolded state and an intermediate state.52 Such conformational change might be involved in the rapid conformational transition (< 10 µs) in the

22


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unfolded-state ensemble indicated by the present 2D-FLCS study. Based on the labeling-position dependence, it was concluded that the nanosecond dynamics observed in the previous study corresponds to the partial formation of all helices.52 Thus, the rapid conformational transition in the unfolded-state ensemble might also involve the partial helix formation/deformation of the three helices. In a previous high-speed single-molecule tracking study, Oikawa et al. observed that the FRET efficiency of the unfolded state is broadly distributed and that the FRET efficiency of a substantial number of single unfolded molecules retain certain values in their observation time window of a few milliseconds.29 This seemed to indicate the presence of different unfolded conformations that last on the time scale longer than a few milliseconds. In the present 2D-FLCS study, however, such long lasting unfolded conformations were not clearly resolved. The reason of this discrepancy is not clear at the moment, but it might be ascribed to the variation of the acceptor fluorescence quantum yield which is caused by local structural heterogeneity around the acceptor dye.29,53 Because 2D FLCS only utilizes the donor fluorescence lifetime, it is unaffected by variation of the acceptor fluorescence quantum yield, whereas a FRET measurement based on the donor/acceptor intensity ratio is sensitive to it. The highly inhomogeneous nature of the native state, which was clearly indicated

23



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by the present 2D-FLCS study, is much more surprising compared to the inhomogeneity of the unfolded state. For gradual change of the native-state structure, there have been a few studies which reported it can change depending on the condition.16-18,54 For example, a study on a mutant from the protein GB1 domain (56 amino acids) indicated that the structure of the native state is quite fragile and its conformation melts with increase of the denaturant concentration.54 Similarly, smFRET measurements on α-spectrin SH3 domain (62 amino acids) clearly showed gradual disordering of the native state depending on the GdmCl concentration.16 The significance of the present study beyond these previous works is that 2D FLCS revealed that, even at one denaturant concentration, the native state has a variety of conformations (i.e. the ensemble nature) and that different conformations in the native-state ensemble are interconverted to each other on the time scale shorter than 10 microseconds. We note that it has been believed that the conformation of the native state of BdpA is robust and does not change with the change in the solvent condition.31 However, the 2D FLCS data obtained for sample 5-55 clearly show the inhomogeneity of the native state as well as gradual melting of its average structure with increase of the GdmCl concentration. The origin of the ensemble nature of the native state can be inferred from the result of sample 22-55. In the sample 22-55, the donor fluorescence of the native state is

24


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highly quenched, implying that the FRET dyes in the native state are located close to each other even at 2.0 M GdmCl condition. This observation accord well with a previous time-resolved FRET study in which FRET dyes were introduced to the same 22 and 55 positions. 26 Although the FRET dyes are different from sample 22-55, the fluorescence lifetime analysis assuming the native state having a static well-defined structure (with the unfolded state) was successfully performed, which also suggests that the distance between the 22 and 55 positions of the native state is independent of the denaturant condition. Because the 22 position corresponds to the loop region between helices 1 and 2, and the position of 55 is located at the C-terminal end of helix 3, the unchanged 22-55 distance implies that the structure between the 22 and 55 positions, i.e., helix 2, helix 3 and the loop between them, is retained and unchanged in the native-state ensemble. This means that the inhomogeneity of the native state of BdpA arises from other part, i.e. helix 1. In fact, this conclusion is consistent with a previous CD study, which showed that the α-helix contents of synthetic peptides corresponding to helix 1 (residues 3-21) and helices 1-2 (residues 3-39) are quite low, compared to the peptides corresponding to helix 3 (residues 37-58) and helices 2-3 (residues 22-58).24 Because a low α-helix content implies low stability of the corresponding helical structure, this CD study implies that helix 1 is most unstable among the three helices.

25



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Furthermore, extensive Φ-value analyses indicated that among the three helices, only helix 2 is formed in the transition state between the native and unfolded states,33,34 suggesting that helix 2 is the most stable in the native-state ensemble. This also accords with our conclusion that helices 2 and 3 are conserved in the native-state ensemble. As for inhomogeneity arising from helix 1, we note that H/D exchange experiments indicated that the N-terminus region is exposed to the solvent, probably due to fast structural fluctuation of this region.24 Therefore, it is very reasonable to think that the structural inhomogeneity of the native state of BdpA mainly stems from the structural fluctuation of the N-terminus region, and that elongation of the average structure of the native-state ensemble arises from gradual melting of α-helix 1 which is induced with increasing GdmCl concentration. It is worth noting that α-spectrin SH3 domain, 62 amino acids β-sheet protein, also showed the gradual melting of the native state.16 This NMR experiment suggested that the breakage of the intramolecular hydrogen bonds and the consequent melting of the β-sheet is the origin of the native-state disordering.16 Our data suggest that the structural inhomogeneity of the native state of BdpA arises from the gradual melting of helix 1. This may imply that disordering of secondary structures due to the breakage of the intramolecular hydrogen bonds is a common origin of the ensemble nature of the native

26


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state for small proteins. At this moment, we are not able to assign each peak in the lifetime distribution to a specific conformation of BdpA because it is difficult to extract accurate lifetime values from multi-exponential decays having more than two components. In fact, we think that the multiple peaks in the lifetime distribution of sp1 and sp1’ likely represent continuous structural distribution of the native- and unfolded-state ensembles, respectively, rather than indicate a small number of distinct conformations and/or different dye configurations. Nevertheless, the average structure of the native- and unfolded-state ensembles of BdpA can be discussed based on the average donor fluorescence lifetime. The average fluorescence lifetime ( τ ) of each ensemble is calculated from the corresponding lifetime distribution (sp1 or sp1’) by,

∑ α (τ )τ ∑ α (τ ) i

τ =

i

i

.

(2)

i

i

Using τ calculated by eq 2, the average FRET efficiency of each ensemble ( E ) is obtainable by the following equation: E = 1 −  τ   τ0 

,

(3)

where τ0 is the fluorescence lifetime of the donor in the absence of acceptor. Then, the calculated

E

can be converted to the average donor-acceptor distance ( r ) by using eq

1. 27



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For the native-state ensemble of sample 5-55,

E

at 1.0 and 3.0 M GdmCl

conditions are calculated as 0.84 and 0.73, which correspond to r = 38.5 and 43.0 Å, respectively. The r value at 1.0 M GdmCl is longer than the expected donor-acceptor distance (30 Å) which is calculated from Cα carbon of the labelled amino-acid residues on the NMR structure shown in Fig. 1. This is consistent with our argument that the native conformation is quite fragile and helix 1 melts (at least partly) even at 1.0 M GdmCl. On the other hand, the

E

value of the unfolded-state ensemble of sample 22-55 is

changed from 0.73 to 0.59 with the change of the GdmCl concentration from 2.0 to 4.0 M. This corresponds to the change from 45.1 to 50.2 Å in r . The calculated r at 4.0 M GdmCl (50.2 Å) is close to that of the unfolded BdpA (52 Å) which is estimated by assuming worm-like chain with the persistence length lp = 11 Å, i.e. the characteristic lp reported for highly denatured proteins.29,55 This suggests that BdpA is almost fully unfolded at 4.0 M GdmCl concentration and it already has a conformation close to that at a higher denaturant concentration. This estimation also suggest that nonspecific diffusive dynamics of the protein backbone is the origin of the ensemble nature of the unfolded state. The folding mechanism of BdpA has been extensively studied with both of the

28


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ensemble-averaged

and

the

single-molecule

experiments

employing

various

combinations of the FRET pair and labeling positions. The dye positions in sample 5-55 is similar to the one used in a previous smFRET study carried out by Huang et al,31 who utilized a mutant in which FRET dyes are attached at residues 10 and 59. The GdmCl-dependence of the FRET efficiency histogram of this mutant also showed the gradual structural change of the unfolded state as observed in the present study. However, their data did not show GdmCl-dependent shifts in the FRET efficiency of the native state which is clearly observed in the present study. (They observed a slight shift, but they attributed it to the change in the fluorescence quantum yield and the refractive index.) We think that the discrepancy between this previous smFRET study and the present 2D-FLCS study is due to the low resolution of smFRET for the FRET efficiency, although the small difference in the dye positions (residues 10 and 59 vs 5 and 55) and/or the additional mutation in our sample (Y15F) might also have some effects. We note that, in our data, the native-state ensemble of sample 5-55 clearly showed a small but significant shift in

E

from 0.84 to 0.73 with increase of the GdmCl concentration.

Such a small shift can hardly be resolved in conventional smFRET measurements in which the width of an individual peak is as large as 0.2 ~ 0.3 in the FRET efficiency histogram. Actually, Oikawa et al. recently achieved a much narrower shot-noise width

29



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(< 0.1) in their line-confocal tracking of smFRET experiments,28,29 and successfully detected a gradual shift of the FRET efficiency of the native-state for sample 5-55.29 This demonstrates the high-resolution in conformational analysis accomplished by 2D FLCS, which makes 2D FLCS a new powerful tool to elucidate the complex conformational dynamics of proteins at the single molecule level with unprecedented time-resolution.

CONCLUSION

In this study, 2D FLCS was performed on B domain of protein A to elucidate its conformational property and dynamics of this small protein in the folding/unfolding process in equilibrium using GdmCl as the denaturant. The obtained results showed that the conformations appearing in the folding/unfolding process are divided into the native and unfolded states, but both are highly heterogeneous. The 2D FLCS data also revealed that the conformational transition in each ensemble occurs on the time scale shorter than 10 microseconds and that the average structures of both ensembles are elongated with increasing the GdmCl concentration. The experiments performed for two FRET mutants suggested that the fraying of helix 1 is most likely the origin of the native-state 30


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inhomogeneity. The conformational flexibility of helix 1 suggested in this study may be relevant to the multiple binding property of protein A.56,57 As already noted, it has been shown that several small proteins exhibit non-cooperative, downhill-like folding processes beyond the simple two-state folding. The present 2D-FLCS study indicates that, even in the case that a protein looks a simple two-state folder, the actual folding/unfolding process contains complex conformational transitions, and hence the ensemble nature of not only the unfolded state but also the native state needs to be taken into account for discussing its folding/unfolding mechanism. In other words, our findings suggest that BdpA has a folding property in between simple two-state and downhill.42 We think that this view obtained for BdpA is likely a rather common feature for many small proteins. Further 2D-FLCS study will clarify such aspects of conformational property and dynamics of small proteins which will be essential for understanding their folding/unfolding processes.

ASSOCIATED CONTENT

31



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Page 32 of 56

Supporting Information. Detailed

description

of

the

global

2D

inverse

Laplace

transform,

GdmCl-dependence of the peak lifetime of sp2, rigorous analysis of the data of sample 22-55, fluorescence correlation curve of sample 5-55, 2D FLCS of sample 5-55, fluorescence spectra of acceptor, 2D FLCS of sample 22-55, comparison of the results between ensemble-averaged data and 2D FLCS data, and elements of gij(∆T). The supporting information is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION

Corresponding Author

* E-mail: [email protected]. Phone: +81 48 467 4592, ext. 8804

Present Address ∇

T.O: Department of Applied Chemistry, Graduate School of Science and Engineering,

Saitama University, 255 Shimo-Okubo, Sakura, Saitama 338-8570, Japan

Author Contributions 32


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This work was partly supported by the RIKEN SPDR Program (to T.O.), MEXT KAKENHI Grant Number JP25104007 (to S.T.), and JSPS KAKENHI (Grant-in-Aid for Scientific Research on Innovative Areas) Grant Number JP25104005.

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Kuzmenkina, E. V.; Heyes, C. D.; Nienhaus, G. U., Single-Molecule Forster

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Figure Captions Figure 1. NMR structure of B domain of protein A (PDB ID: 1SS1). The residues which were mutated to cysteine are shown by sticks.

Figure 2. (A) Fluorescence spectra in K5C/Y15F/A55C mutant (sample 5-55) of B domain of protein A at various concentrations of guanidium chloride. Excitation wavelength was set at 480 nm. (B) Fluorescence decay curves of donor in K5C/Y15F/A55C mutant (sample 5-55) of B domain of protein A at various concentrations of guanidium chloride. The decay curves are normalized for the fluorescence intensity at t = 0. Excitation wavelength was set at 483 nm, and donor fluorescence was selectively detected by using a bandpass filter (525/45 nm).

Figure 3. Two-dimensional emission-delay correlation maps (A, B), two-dimensional lifetime correlation maps (C, D) and the independent lifetime distributions (E) obtained for K5C/Y15F/A55C mutant (sample 5-55) of B domain of protein A in the presence of 2.0 M guanidium chloride. The two-dimensional lifetime correlation maps and independent lifetime distributions were obtained by the global 2D-MEM analysis of the two-dimensional emission-delay correlation maps obtained at ∆T = 1 ~ 20 µs, 50 ~ 100

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µs and 400 ~ 600 µs, and only the data at ∆T = 1 ~ 20 µs (A, C) and 400 ~ 600 µs (B, D) are shown.

Figure 4. Two-dimensional fluorescence lifetime correlation maps (A-C) and the corresponding independent lifetime distributions (D-F) of K5C/Y15F/A55C mutant (sample 5-55) of B domain of protein A in the presence of 1.0 M (A, D), 2.5 M (B, E) and 3.0 M (C, F) guanidium chloride. They were obtained by the global 2D-MEM analysis of two-dimensional emission-delay correlation maps obtained at ∆T = 1 ~ 20 µs, 50 ~ 100 µs and 400 ~ 600 µs and only two-dimensional fluorescence lifetime correlation maps at ∆T = 1 ~ 20 µs are shown.

Figure 5. Fluorescence decay curves corresponding to sp1 at various concentrations of guanidium chloride. These curves were obtained by performing Laplace transform on the lifetime distribution of sp1 shown in Figs. 3 and 4. The data are normalized at the intensity at t = 0.

Figure 6. (A) Fluorescence spectra in Y15F/N22C/A55C mutant (sample 22-55) of B domain of protein A at various concentrations of guanidium chloride. Excitation

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wavelength was set at 488 nm. (B) Fluorescence decay curves of donor in Y15F/N22C/A55C mutant (sample 22-55) of B domain of protein A at various concentrations of guanidium chloride. Each trace is shown with the respective colors indicated in the panel A. Excitation wavelength was set at 483 nm, and donor fluorescence was selectively detected by using a bandpass filter (525/45 nm). The data are normalized by the fluorescence intensity at the corresponding wavelength regions shown in (A). The data normalized by the intensity at t = 0 (ns) are also shown in the inset.

Figure 7. Two-dimensional lifetime correlation maps (A, B) and the corresponding independent lifetime distributions (C, D) of Y15F/N22C/A55C mutant (sample 22-55) of B domain of protein A at 2.0 M (A, C) and 4.0 M (B, D) guanidium chloride conditions. The data were obtained by the global 2D-MEM analysis of two-dimensional emission-delay correlation maps calculated for ∆T = 1 ~ 20 µs, 50 ~ 100 µs and 400 ~ 600 µs, and only the data at ∆T = 1 ~ 20 µs are shown. In C and D, the relative amplitudes of sp1’ and sp2’ on the corresponding two-dimensional lifetime correlation maps (A and B) are given in parentheses. (E) Fluorescence decay curves of sp1’ at 2.0 M (red) and 4.0 M (blue) guanidium chloride conditions. These decay curves were

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obtained by Laplace transform of the lifetime distributions of sp1’s shown in C and D. The data are normalized by the intensity at t = 0.

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Table 1. Relative Contribution of Shorter (sp1) and Longer (sp2) Independent Lifetime Distributions on the Two-dimensional Lifetime Correlation Maps.

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TOC GRAPHIC

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Fig1: the structure of BdpA 100x53mm (300 x 300 DPI)



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Fig2: fluorescence spectra and fluorescence decay curves of sample 5-55 100x162mm (300 x 300 DPI)


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Fig3: 2D FLCS of sample 5-55 at 2M guanidium chloride 212x137mm (300 x 300 DPI)



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Fig4: 2D FLCS of sample 5-55 at 1M, 2.5M, and 3M guanidium chloride 204x123mm (300 x 300 DPI)


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Fig5: fluorescence decay curves of sp1 93x75mm (300 x 300 DPI)



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Fig6: fluorescence spectra and fluorescence decay curves of sample 22-55 101x161mm (300 x 300 DPI)


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Fig7: 2D FLCS of sample 22-55 at 2M and 4M guanidium chloride 207x138mm (300 x 300 DPI)



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Table1: relative contribution of shorter (sp1) and longer (sp2) independent lifetime disctributions 68x23mm (300 x 300 DPI)


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Highly Heterogeneous Nature of the Native and ... - ACS Publications

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