Microsecond Subdomain Folding in Dihydrofolate Reductase

doi:10.1016/j.jmb.2011.04.057

J. Mol. Biol. (2011) 410, 329–342 Contents lists available at www.sciencedirect.com

Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

Microsecond Subdomain Folding in Dihydrofolate Reductase Munehito Arai 1,2 ⁎, Masahiro Iwakura 1 , C. Robert Matthews 3 and Osman Bilsel 3 ⁎ 1

Protein Design Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan 2 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan 3 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA Received 22 December 2010; received in revised form 19 March 2011; accepted 21 April 2011 Available online 30 April 2011 Edited by F. Schmid Keywords: protein folding; folding intermediate; microsecond mixing; FRET; fluorescence lifetime

The characterization of microsecond dynamics in the folding of multisubdomain proteins has been a major challenge in understanding their often complex folding mechanisms. Using a continuous-flow mixing device coupled with fluorescence lifetime detection, we report the microsecond folding dynamics of dihydrofolate reductase (DHFR), a two-subdomain α/ β/α sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Förster resonance energy transfer, and the dynamic properties of the local Trp environments were monitored by fluorescence lifetime detection. We found that substantial collapse occurs in both the locally connected adenosine binding subdomain and the discontinuous loop subdomain within 35 μs of initiation of folding from the urea unfolded state. During the fastest observable ∼ 550 μs phase, the discontinuous loop subdomain further contracts, concomitant with the burial of Trp residue(s), as both subdomains achieve a similar degree of compactness. Taken together with previous studies in the millisecond time range, a hierarchical assembly of DHFR—in which each subdomain independently folds, subsequently docks, and then anneals into the native conformation after an initial heterogeneous global collapse— emerges. The progressive acquisition of structure, beginning with a continuously connected subdomain and spreading to distal regions, shows that chain entropy is a significant organizing principle in the folding of multisubdomain proteins and single-domain proteins. Subdomain folding also provides a rationale for the complex kinetics often observed. © 2011 Elsevier Ltd. All rights reserved.

*Corresponding authors. M. Arai is to be contacted at Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan. E-mail addresses: [email protected]; [email protected]. Abbreviations used: DHFR, dihydrofolate reductase; DLD, discontinuous loop subdomain (residues 1−37 and 107−159); ABD, adenosine binding subdomain (residues 38−106); CF, continuous flow; SAXS, small-angle X-ray scattering; FRET, Förster resonance energy transfer; IAEDANS, 5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid; AEDANS, 5-(((acetylamino)ethyl)amino)naphthalene-1-sulfonate; TCSPC, time-correlated single-photon counting. 0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.

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Introduction The elucidation of protein folding mechanisms remains an outstanding challenge in molecular biology. The folding mechanisms of small singledomain proteins with fewer than 100 residues have been extensively studied experimentally and theoretically. Very often, these small proteins and domains fold via a single-exponential process in the microsecond time range whose rate constant reflects the chain topology.1 Proteins with a higher proportion of local contacts fold faster than those with a higher proportion of nonlocal contacts in the native conformation.1 With advances in computational power, it is now becoming possible to predict the folding processes2,3 and the final three-dimensional structures of small proteins.4 By contrast, the folding mechanisms of large multi-subdomain proteins with more than 100 residues remain to be solved. Such proteins often fold in the submillisecond time range to molten globule-like compact intermediates with pronounced secondary structure but little defined tertiary structure. Subsequently, they acquire specific side-chain packing interactions and the native structure in the milliseconds-to-seconds timescales.5–7 Direct measurement of the microsecond folding dynamics of large proteins by a variety of probes is required to understand the initial biases and very short-lived high-energy partially folded states that guide the eventual formation of the functional native state. Dihydrofolate reductase (DHFR) from Escherichia coli is a monomeric 159-residue α/β/α protein consisting of the discontinuous loop subdomain (DLD; residues 1–37 and 107–159) and the adenosine binding subdomain (ABD; residues 38–106).8 It catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate and has been a target enzyme of antifolate drugs.9,10 Moreover, DHFR has served as a paradigm for studying the relationship between the structure, function, and dynamics of enzymatic transformations.11,12 Relevant to the present study, DHFR has been the target of numerous studies of its folding reaction. Within the first few milliseconds, DHFR accumulates a kinetic folding intermediate (termed I5) in which ∼25% of the far-UV circular dichroism (CD) signal is developed13–16 and two subsets of hydrogen-bonding networks, corresponding to two hydrophobic clusters, are formed.17 In the subsequent folding phase (τ5 phase), DHFR forms a set of four hyperfluorescent intermediates (I1–I4) with a time constant of 200 ms.18,19 These intermediates then fold into a corresponding set of native or native-like conformers through four parallel folding channels (τ1–τ4 phases).18,19 Recently, we have shown, using a continuousflow (CF) small-angle X-ray scattering (SAXS) technique,20,21 that microsecond hydrophobic col-

Microsecond Subdomain Folding in DHFR

lapse occurs in the folding of DHFR within the dead time of 300 μs.22 Here, we extend the time resolution by ∼ 10-fold using a CF mixing device coupled with fluorescence lifetime detection and time-resolved Förster resonance energy transfer (FRET).23 With these techniques, we were able to qualitatively probe the global geometrical dimensions and local Trp environments of DHFR after the first 35 μs of folding. We find heterogeneous global collapse within 35 μs and subsequent subdomain structure formation in the submillisecond time range.

Results Design and characterization of mutants Although intramolecular distance information can be obtained by FRET from intrinsic Trp donors to an acceptor, 5-({2-[(iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid (IAEDANS),24 the goal of the present study is to use FRET in a qualitative manner to probe the organization of the DLD and the ABD and to delineate their roles in the global folding of DHFR. The focus on the qualitative interpretation of the FRET experiments reflects the fact that E. coli DHFR contains five tryptophans: Trp22, Trp30, and Trp133 in the DLD, and Trp47 and Trp74 in the ABD (Fig. 1a). As will be shown below, although individual Trp residues can be replaced with other amino acids, the destabilization accompanying the simultaneous replacement of multiple Trp residues precludes FRET studies of single-donor DHFR variants. An IAEDANS label was introduced individually at Arg52, Asp87, and Gln146, which are located in the ABD, intersubdomain region, and DLD, respectively (Fig. 1a). These positions for the 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonate (AEDANS) acceptors were selected because they are less well conserved, protrude into the solvent, and are not involved in α-helices, β-strands, or other folding elements that are essential for the protein to fold. 26 These positions, by being close in sequence or close in space to Trp donors, highlight the dimensional properties of the ABD, the DLD, and their interface. As expected, the overall structure (examined by CD spectra) and the stability (examined by ureainduced equilibrium unfolding) of the R52C, D87C, and Q146C mutants and their IAEDANSlabeled forms (denoted as R52AEDANS, D87AEDANS, and Q146AEDANS, respectively) were coincident with those of AS-DHFR, a C85A/ C152S Cys-free mutant of DHFR, which has been used as a pseudo-wild-type version of DHFR22,27 (Fig. 2a–c, Table 1). The steady-state FRET efficiencies of the R52AEDANS, D87AEDANS, and Q146AEDANS mutants in the native state were

331

Microsecond Subdomain Folding in DHFR

the τ5 phase, in which side-chain packing interactions between Trp47 and Trp74 are formed, may be due to the attachment of AEDANS at Asp87 in close proximity to the Trp residues, affecting the formation of such interactions. However, D87AEDANS will not affect the folding reaction before the τ5 phase, which we focus on in this study. Contribution of each Trp residue to the fluorescence spectrum The five intrinsic Trp residues of DHFR preclude a residue-specific quantitative interpretation of the FRET data. To estimate a fractional contribution of each Trp residue to the fluorescence spectrum of DHFR, we replaced individual Trp residues of the wild-type DHFR with either Leu or Tyr. Although the mutations decreased both fluorescence intensity and stability, the variant proteins formed stably folded structures under native conditions (Table 2). A resolved fluorescence spectrum of each Trp residue, calculated by subtracting the spectrum of a variant with a single Trp substitution from that of the wild-type protein, showed that Trp74 has a dominant contribution to the overall fluorescence intensity of native DHFR (Fig. 1b). The sum of the individual resolved spectra was coincident with the fluorescence spectrum of the wild-type DHFR (Fig. 1b), suggesting that intramolecular interactions do not significantly perturb the spectra and quantum yields of the individual Trp residues. From the areas of the resolved spectra, the fractional contributions of individual Trp residues to the fluorescence intensity of native DHFR were estimated to be 13% (Trp22), 17% (Trp30), 9% (Trp47), 49% (Trp74), and 12% (Trp133), demonstrating that Trp74 contributes nearly half of the fluorescence intensity of native DHFR (Table 3). Fig. 1. (a) A representation of DHFR (Protein Data Bank ID: 1rx1). The five Trp residues and three mutation sites for acceptor placement are shown by sticks and spacefilling, respectively. The DLD and the ABD are shown in magenta and cyan, respectively. β-Strands A, F, G, and H are labeled. The figure was drawn using MOLMOL.25 (b) Resolved fluorescence spectra obtained by subtracting the fluorescence spectrum of a single Trp substitution mutant from that of the wild-type protein. Color codes are shown in the panel. The sum of the resolved fluorescence spectra and the spectrum of the wild-type DHFR are shown by red dotted and black continuous lines, respectively.

0.66, 0.46, and 0.41, respectively (Fig. 2d). Kinetic folding measurements using stopped-flow and manual-mixing devices equipped with steady-state total intensity fluorescence detection showed that the mutations and IAEDANS labels only slightly affected the folding relaxation times of the τ5–τ1 phases (Fig. S1 and Tables S1 and S2 in Supplementary Data). The slower folding of D87AEDANS in

Equilibrium unfolding of AS-DHFR The urea-induced equilibrium unfolding transition of unlabeled AS-DHFR was measured by timeresolved Trp fluorescence using time-correlated single-photon counting (TCSPC). The fluorescence decay curves were well fit to a three-exponential function at all urea concentrations (average reduced χ2 = 1.04) (Fig. S2) and confirmed by analyses via the maximum entropy method.30 The three lifetimes at 0 M urea were 5.77 ± 0.01 ns, 1.79 ± 0.03 ns, and 0.51 ± 0.01 ns. Assignment of each lifetime to a single Trp residue is, however, not possible because even a single-Trp protein has multiple lifetimes that reflect the heterogeneity of local Trp environments. 31 Instead, we used an average excited-state lifetime, which is independent of the number of lifetimes,32 as a metric for probing folding. Consistent with steadystate fluorescence measurements (Table 1), an intensity averaged lifetime, τave (see Eq. (1) in

332

Microsecond Subdomain Folding in DHFR

Fig. 2. (a and b) CD spectra of the unlabeled (a) and IAEDANS-labeled (b) mutants of AS-DHFR. The spectrum of ASDHFR is shown in both panels. (c) Normalized equilibrium unfolding transition curves of the unlabeled and IAEDANSlabeled mutants of AS-DHFR. The dotted and continuous lines are fitting curves to a two-state transition. The thermodynamic parameters are listed in Table 1. (d) Fluorescence spectra of the unlabeled (dotted lines) and IAEDANSlabeled mutants (continuous lines) of AS-DHFR (excitation wavelength, 295 nm). The fluorescence of IAEDANS at ∼ 490 nm is due to FRET from Trp donors. Color codes are shown in each panel. Conditions: 10 mM potassium phosphate (pH 7.8), 0.2 mM K2EDTA, 1 mM 2-mercaptoethanol, and 2 μM protein at 23 °C.

Materials and Methods), shows a sigmoidal transition (Fig. 3a). The higher value of τave under native conditions is likely due to the burial of Trp side chains in the native state.32 The gradual decrease in τave in the unfolded baseline as the urea concentration is decreased might reflect the increasing potency of the solvent in the dynamic quenching of Trp fluorescence at lower concentrations of urea, where a lower solvent viscosity accelerates the binding of water to an indole ring.32 Kinetic folding of AS-DHFR by CF-TCSPC The changes in the Trp fluorescence lifetimes of AS-DHFR during folding after a 4.5 to 0.45 M urea concentration jump were monitored using a microsecond-resolved CF mixing device coupled with fluorescence lifetime detection (TCSPC).23,33 The measurement of fluorescence lifetimes at each point increases the robustness of the FRET measurement because, for a monomeric system, the

lifetime is independent of protein concentration and also independent of incident excitation power under the conditions of our experiment. The Trp fluorescence decay curves at various folding time points were fit to a two-exponential function because the short lifetimes were not resolved due to lower signal-to-noise ratios resulting from the shorter acquisition times in the CF-TCSPC experiment (Fig. S3a). The kinetic progress curves of the Trp lifetimes and amplitudes along the folding time axis were globally fit to a single-exponential function, resulting in a common folding relaxation time of 550 ± 110 μs (Fig. S3). The kinetic folding curve represented by τave also fit well to a single exponential with this time constant. The τave values extrapolated to zero time of the folding reaction and, after this exponential phase, were 3.57 ± 0.02 ns and 3.81 ± 0.03 ns, respectively (Fig. 3a and b). When the folding reaction was measured at 2 M urea, the lifetimes, amplitudes, and τave values did not change between 35 μs and 800 μs, and the

333

Microsecond Subdomain Folding in DHFR

Table 1. Thermodynamic parameters of AS-DHFR and its mutants Protein

Method

ΔG0 (kcal mol− 1)

m (kcal mol− 1 M− 1)

cM (M)

ΔΔG (kcal mol− 1)a

ΔΔGlabel (kcal mol− 1)b

ssFLc ssFL ssFL ssFL ssFL ssFL ssFL trFLf trFL trFL trFL

4.9 ± 0.1d 6.25 ± 0.08 6.6 ± 0.1 5.0 ± 0.1 4.6 ± 0.1 5.5 ± 0.1 4.8 ± 0.1 3.8 ± 0.1 6.3 ± 0.2 7±2 5.0 ± 0.2

1.80 ± 0.04 2.22 ± 0.03 2.13 ± 0.03 2.10 ± 0.04 1.60 ± 0.03 2.10 ± 0.04 1.59 ± 0.04 1.43 ± 0.03 2.17 ± 0.06 3.3 ± 0.8 1.92 ± 0.07

2.70 ± 0.01 2.81 ± 0.01 3.10 ± 0.01 2.40 ± 0.01 2.86 ± 0.01 2.64 ± 0.01 3.01 ± 0.01 2.64 ± 0.01 2.90 ± 0.01 2.27 ± 0.06 2.62 ± 0.02

— 0.21 ± 0.03e 0.78 ± 0.03 − 0.59 ± 0.03 0.27 ± 0.02 − 0.12 ± 0.03 0.51 ± 0.03 — 0.47 ± 0.03 − 0.9 ± 0.2 − 0.03 ± 0.04

— 0.63 ± 0.03

AS-DHFR R52C R52AEDANS D87C D87AEDANS Q146C Q146AEDANS AS-DHFR R52AEDANS D87AEDANS Q146AEDANS

0.85 ± 0.03 0.67 ± 0.03 NDg ND ND ND

All measurements were recorded at 23 °C in 10 mM potassium phosphate buffer (pH 7.8) with 0.2 mM K2EDTA and 1 mM 2mercaptoethanol. a The difference in ΔG between AS-DHFR and the mutant (ΔΔG) was estimated at the mean value of their cM values (see Eq. (5)). b ΔΔGlabel is the difference in ΔG between labeled proteins and unlabeled proteins. c The data were obtained from steady-state Trp fluorescence measurements. d Errors are the standard errors of fitting. e Errors for ΔΔG and ΔΔGlabel were calculated by propagation of uncertainties. f The data were obtained from time-resolved Trp fluorescence measurement in terms of τave. g Not determined.

average of τave values during this time range was 3.50 ± 0.01 ns (Fig. 3b; Fig. S3). Comparison between equilibrium data and kinetic data in terms of τave indicates that within 35 μs of folding, Trp residues are, on average, in local environments similar to those of the unfolded state (Fig. 3a). The τave at 0.45 M urea is slightly larger than that expected of a linear extrapolation of the unfolded-state baseline, suggesting that Trp residues are partly buried in the protein molecule within 35 μs but are largely exposed to solvent. Local Trp environments change during the 550 μs

Table 2. Thermodynamic parameters of single Trp substitution mutants Protein Wild typeb W22Ld W30Yb W47Lf W74Lf W133Y

ΔG0 (kcal mol− 1)

m (kcal mol− 1 M− 1)

cM (M)

ΔΔGa (kcal mol− 1)

6.3 ± 0.3c

1.97 ± 0.09

3.18 ± 0.02

—

6.2 ± 0.2 4.1 ± 0.2 5.6 ± 0.2 4.0 ± 0.2 3.5 ± 0.3

2.38 ± 0.08 1.75 ± 0.07 2.10 ± 0.07 2.32 ± 0.09 1.7 ± 0.1

2.6 ± 0.1 2.35 ± 0.02 2.7 ± 0.1 1.7 ± 0.1 1.99 ± 0.04

− 1.2 ± 0.2e −1.54 ± 0.07 −1.0 ± 0.2 −3.1 ± 0.2 −2.2 ± 0.1

All measurements were recorded at 23 °C in 10 mM potassium phosphate buffer (pH 7.8) with 0.2 mM K2EDTA and 1 mM 2mercaptoethanol. a ΔΔG is the free-energy difference between the mutant and the wild-type DHFR. b Thermodynamic parameters were taken from Arai and Iwakura.16 c The errors are the standard errors of fitting. d Thermodynamic parameters were taken from Arai et al.15 e Errors for ΔΔG were calculated by propagation of uncertainties. f Thermodynamic parameters were taken from Ohmae et al.28

phase, in which a “burst-phase” intermediate observed by stopped-flow experiments is formed.34 It should be noted that when an amplitude averaged lifetime, 〈τ〉 (see Eq. (3) in Materials and Methods), was used instead of the intensity averaged lifetime τave, the same conclusions were obtained (see Fig. S4). The amplitude averaged lifetime 〈τ〉 is preferred in calculating FRET efficiencies because it rigorously tracks mole fraction.35 The intensity averaged lifetime (i.e., the first moment of the decay) is also used, since a nonlinear least squares analysis is not required and, for complex excited-state decays, can provide greater precision. τave is a good approximation to the more rigorous parameter 〈τ〉 when the quantum yields of all species are comparable. Equilibrium unfolding of IAEDANS-labeled mutants Time-resolved Trp fluorescence decay curves of the IAEDANS-labeled variants had four lifetimes (Fig. S5), confirmed by exponential fitting and maximum entropy analyses. In the unfolded state, the three longer lifetimes were the same as those of unlabeled AS-DHFR. However, the presence of FRET from Trp donors to an IAEDANS acceptor shortened the long Trp lifetime in the native state and caused the appearance of an additional fast lifetime (∼ 0.2 ns). As a result, the amplitude averaged lifetime 〈τ〉, which was used to calculate FRET efficiency (see Eqs. (2) and (3) in Materials and Methods), was decreased for the labeled mutants, compared with that of the unlabeled protein throughout the unfolding transition (Fig. 4a). Although the 〈τ〉 values and FRET efficiencies were

334

Microsecond Subdomain Folding in DHFR

Table 3. Cβ–Cβ distance between Trp residues and mutation sites Trp Trp22 Trp30 Trp47 Trp74 Trp133

f(QY)a (%)

ASA (N/U)b (%)

Arg52c [Å (%)]

13 17 9 49 12

14 8 8 23 5

21.4 ± 0.8 (54) 20.0 ± 0.5 (64) 9.5 ± 0.2 (99) 15.4 ± 0.2 (89) 32.7 ± 0.2 (8.6)

Asp87c [Å (%)] 36.5 ± 0.5 26.2 ± 0.2 24.2 ± 0.1 20.6 ± 0.1 25.8 ± 0.3

(4.6) (26) (36) (60) (28)

Gln146c [Å (%)] 11.7 ± 0.4 (98) 13.0 ± 0.3 (96) 27.0 ± 0.7 (23) 35.5 ± 0.6 (5.4) 29.8 ± 0.3 (14)

Twelve crystal structures with Protein Data Bank IDs 1rx1, 1rx2, 1rx3, 1rx4, 1rx5, 1rx6, 1ra9, 4dfr_A, 4dfr_B, 5dfr, 6dfr, and 7dfr were used to calculate the distance between the Cβ atom of Trp and the Cβ atom of the mutation site at which IAEDANS was introduced. The mean and standard deviation of the Cβ–Cβ distances are shown. a Fractional contribution of the corresponding Trp residue to the quantum yield of DHFR obtained from Fig. 1b (see the text). b The ratio between the solvent-accessible surface area of the native state of DHFR and the solvent-accessible surface area of the unfolded state of DHFR (Protein Data Bank ID: 1rx1) calculated in accordance with Richmond.29 c The average FRET efficiency values in parentheses were calculated by explicitly considering the donor excited-state rate distribution and FRET rate distribution. The calculation of the average FRET efficiency assumed a Förster distance of 22 Å, a Gaussian donor–acceptor distance distribution centered at the reported crystal structure distance with a full width at half-maximum of 2 Å. A donor excited-state decay distribution with a full width at half-maximum of 0.3 ns− 1 centered at 0.3 ns− 1 was used. The FRET efficiency calculations assume an orientation factor corresponding to full rotational averaging of the donor and the acceptor (κ2 = 2/3). The software for this calculation is available (www.osmanbilsel.net).

similar for all of the labeled mutants in the unfolded state, they differed under native conditions (Figs. 4a and 5a), reflecting the varying proximity of tryptophans to each label (Fig. 1a). Kinetic folding of IAEDANS-labeled mutants by CF-TCSPC Attachment of IAEDANS to AS-DHFR significantly reduced the average lifetime of Trp donors at the earliest stages of folding. The 〈τ〉 value of the R52AEDANS mutant averaged within 100 μs of the folding is 1.32 ± 0.01 ns, which is intermediary between the value of the native state (0.88 ± 0.01 ns) and the value of the unfolded state (1.84 ± 0.02 ns) (Fig. 4a and b). The initial 〈τ〉 value of the D87AEDANS variant is very similar to that of the corresponding native state (Fig. 4c). The 〈τ〉 value for Q146AEDANS decreases from a value similar to the unfolded state to a value similar to the native state over the 100 to 800 μs time range, which was well fit to a relaxation time constant of 550 μs (Fig. 4d). The 〈τ〉 values extrapolated to zero time of the folding reaction and, after this phase, were 1.93 ± 0.02 ns and 1.73 ± 0.01 ns, respectively. FRET efficiencies averaged within 100 μs of folding were 0.53 ± 0.01 and 0.47 ± 0.02 for the R52AEDANS and D87AEDANS mutants, respectively, corresponding to 56 ± 2% and 78 ± 6% of the total change expected between the native state and the unfolded state (Fig. 5a–c). These results indicate a substantial compaction of DHFR around Arg52, Asp87, and Gln146 early in the folding. Subsequently, the 〈τ〉 values of R52AEDANS and D87AEDANS were almost unchanged up to 800 μs, while the 〈τ〉 value of Q146AEDANS showed a definitive decrease (Fig. 4b–d). In terms of FRET efficiency, only the Q146AEDANS mutant showed a slight increase in the microsecond timescale,

which was well fit to a relaxation time constant of 550 μs (Fig. 5d). The FRET efficiencies extrapolated to zero time of the folding reaction and, after this phase, were 0.31 ± 0.01 and 0.41 ± 0.01, corresponding to 33 ± 3% and 76 ± 1%, respectively, of the total expected change from the unfolded state to the native state (Fig. 5a and d). Thus, further compaction around Gln146 occurs in the 550-μs phase. To test the stability of the rapidly formed intermediates, we performed refolding at a higher final urea concentration within the native baseline. Folding at 2 M urea similarly showed that 〈τ〉 was significantly reduced within 35 μs relative to the unfolded baseline extrapolation and was unchanged out to 800 μs (Fig. 4a; Fig. S6). Unlike Q146AEDANS, the FRET efficiency of R52AEDANS was smaller than at 0.45 M (Fig. 5a; Fig. S6), providing insights into the subdomain nature of the folding reaction (see Discussion).

Discussion DHFR folding studies using CF fluorescence lifetime methods extend the time limit of observation to 35 μs, representing an ∼ 10-fold improvement in time resolution compared to our previous study using SAXS as probe.22 In addition to the τ5–τ1 folding phases of DHFR observed by conventional stopped-flow total intensity fluorescence,19 we have found two additional phases: a burst phase (b35 μs), in which substantial compaction of the protein occurs rapidly, and the τ6 phase (∼ 550 μs), in which Trp residues are locally sequestered from solvent concomitant with further compaction around Gln146. Although the presence of five Trp donors limits a full quantitative interpretation of the FRET data, important qualitative insights on the

Microsecond Subdomain Folding in DHFR

Fig. 3. (a) Urea concentration dependence of the τave of AS-DHFR. Black continuous line shows the fitting curve to a two-state transition. Cyan continuous and broken lines show the baselines of the native and unfolded states, respectively. (b) Kinetic folding curves of AS-DHFR monitored by CF time-resolved Trp fluorescence at 0.45 M (red) and 2 M urea (blue). The red continuous line is the fitting curve to a single-exponential function with a relaxation time of 550 μs. (a) shows the τave values extrapolated to zero time of the folding reaction (red filled square) and the τave values after this phase has been completed (red open square). The blue continuous line in (b) is the average of the data points measured at 2 M urea, which is plotted in (a) with a blue filled square.

timescales of subdomain organization and interaction emerge. Details on the structures of intermediates, the folding processes in the microsecond time range, and the folding mechanisms of α/β-type proteins are discussed below. Interpretation of FRET data Dye-labeled Cys variants at Asp87, Gln146, and Arg52 in the ABD, DLD, and intersubdomain region, respectively, can provide insights into the acquisition of structure in this two-subdomain protein. Although the presence of five Trp donors in DHFR complicates residue-specific interpretation

335 of the FRET data, some simplification can be obtained by a consideration of the fractional contribution of each Trp residue to the total fluorescence intensity of DHFR, the spatial distance in the native state between each Trp and the labeled sites, and the amino acid sequence separation distance between the donor–acceptor pair (Fig. 1a, Tables 3 and 4). For example, Trp74, with the highest quantum yield in the native state, is most likely a dominant donor for the D87AEDANS acceptor in both the native state and the denatured state. The Cβ–Cβ Trp74-Asp87 distance in the native structure is 20.6 Å (Table 3) and is the only Trp-D87 distance less than the 22 Å Förster distance. The sequence separation of 13 residues would place this pair in closer proximity than with any other Trp donor in the unfolded state (Table 4). Similarly, Trp22 and Trp30, the second and third most fluorescent donors, are probably dominant donors for Q146AEDANS in the native state. Thus, the D87AEDANS and Q146AEDANS data are considered to reflect primarily the 74–87 FRET pair and the 22–146 and 30–146 FRET pairs that probe the structural events in the ABD and DLD subdomains, respectively. Because Arg52 is close to Trp22, Trp30, Trp47, and Trp74 both in sequence and in space, the FRET data of R52AEDANS are considered to represent the structural events in the intersubdomain region (Tables 3 and 4). In the following, we interpret the present data qualitatively assuming these assignments. The qualitative interpretation outlined above further assumes that the FRET orientation factor does not change significantly in the submillisecond time regime. Stopped-flow time-resolved anisotropy studies by Jones et al.—which showed that the rotational correlation time and fundamental anisotropy of the tryptophans in DHFR measured at 20 ms during refolding are similar to those of the unfolded state under denaturing conditions—support the argument that the orientation factor is not the dominant contributor to the changes in FRET efficiency.34 Given the average anisotropy of the Trp residues (0.038), a change in the steady-state anisotropy of AEDANS from 0.01 to 0.1 would result in a change in the minimum and maximum 2 2 = 0.49 and κmax = 1.2 to values of κ2 from κmin 2 2 κmin = 0.35 and κmax = 1.7, respectively. This translates into an ∼ 1 Å change in the minimum and maximum values of the Förster distance. Although contributions from a change in the orientation factor cannot be ruled out completely, an effect of this magnitude would need to be accompanied by a significant restriction in acceptor mobility. Previous CF-SAXS data, however, indicate relatively minor changes in the global dimensions of DHFR in the submillisecond time range, consistent with minor changes in acceptor conformational dynamics.

336

Microsecond Subdomain Folding in DHFR

Fig. 4. (a) Urea concentration dependence of the 〈τ〉 of R52AEDANS (green), D87AEDANS (red), and Q146AEDANS mutants (blue), and unlabeled wild type (gray). Thick continuous lines show the fitting curves to a two-state transition. Thin broken lines show the baseline of the unfolded state. Filled squares show the initial 〈τ〉 values averaged within 100 μs of the folding reaction. (b–d) Kinetic folding curves of the R52AEDANS (b), D87AEDANS (c), and Q146AEDANS mutants (d) monitored by 〈τ〉 at 0.45 M urea. Continuous and broken lines show the 〈τ〉 values in the native and unfolded states, respectively.

Structures formed within 35 μs Time-resolved Trp fluorescence measurements demonstrate substantial and heterogeneous compaction of the DHFR molecule within 35 μs. The total changes in FRET efficiency between the unfolded and native state were 56 ± 2%, 78 ± 6%, and 33 ± 3% for R52AEDANS, D87AEDANS, and Q146AEDANS, indicating a global contraction of the DHFR molecule. Because the only kinetic phase completed within the 300 μs dead time of the CF-SAXS experiment was the b 35 μs burst phase, the radius of gyration Rg at 35 μs is considered to be the same as that observed at 300 μs (23 Å).22 For comparison, the Rg of the urea-unfolded state extrapolated to 0.45 M urea is 30 Å, and the Rg of the native state is 16.6 Å.22 The structure of the compact folding intermediate formed within 35 μs, termed the I6 intermediate, is heterogeneous: the data suggest differences in the compactness and extent of tertiary contact formation in the ABD relative to the DLD. For D87AEDANS, 78% of the total changes in FRET efficiency between

the unfolded state and the native state were recovered within 35 μs (Fig. 5a and c), indicating that the ABD approaches native-like compactness. A previous pulsed hydrogen-exchange NMR experiment of DHFR found protection of amide hydrogens against solvent exchange for Val75, Ala81, and Ile91 within 5 ms of the folding reaction,17 consistent with the early formation of structure around Trp74 and Asp87 in the ABD. By contrast, the 30% recovery of the FRET efficiency of Q146AEDANS indicates that the DLD is less organized than the ABD. The local connectivity between the antiparallel β-strand pair G-H (spanning residues 132−158) may predispose the β-turn to form rapidly3,38–40 and may partially organize this region of the sheet. However, the full development of the DLD would require the correct registration and docking of the preceding F strand and N-terminal A strand (Fig. 1a). The discontinuous nature of the DLD, with contributions from the N-terminus and the C-terminus and with the possible restrictions in movement engendered by their attachment to the more highly folded ABD, may impede access to a more compact native-like

Microsecond Subdomain Folding in DHFR

337

Fig. 5. (a) Urea concentration dependence of the FRET efficiency of the R52AEDANS (green), D87AEDANS (red), and Q146AEDANS mutants (blue). Thick continuous lines show the fitting curves to a two-state transition. Broken lines show the values in the unfolded state. For Q146AEDANS at 0.45 M urea, filled and open squares show the values extrapolated to zero time of the folding reaction and the values after the τ6 (∼ 550 μs) phase, respectively. Other filled squares show the initial FRET efficiencies averaged within 100 μs of the folding reaction. (b–d) Kinetic folding curves of the R52AEDANS (b), D87AEDANS (c), and Q146AEDANS mutants (d) monitored by FRET efficiency at 0.45 M urea. Continuous and broken lines show the values in the native and unfolded states, respectively. In (d), the thick continuous line is the fitting curve to a single-exponential function with a relaxation time of 550 μs.

structure. The intermediate (56 ± 2%) recovery of the native FRET efficiency for R52AEDANS could be understood in terms of native-like FRET for the Trp residues in the ABD (Trp47 and Trp74) and a diminished contribution from the Trp residues in the less well-folded DLD (Trp22, Trp30, and Trp133). Throughout the first few hundred microseconds of folding, the Trp residues are largely exposed to solvent and susceptible to dynamic quenching by water. Taken together, the I6 intermediate, formed within 35 μs of the folding of DHFR, has substantially compact and heterogeneous structures in which the ABD is significantly contracted but the DLD is less ordered. The τ6 (∼ 550 μs) phase Following the formation of the I6 intermediate, the DLD attains the majority of the overall change in FRET efficiency in the 550 μs kinetic phase. The

Q146AEDANS-labeled DHFR shows an increase in FRET efficiency from 0.31 ± 0.01 to 0.41 ± 0.01, which corresponds to a 33 ± 3% to 76 ± 1% recovery of the overall change. This indicates that the average distance between Trp22/Trp30 and Gln146 becomes more native-like in the τ6 phase, and that the DLD becomes as compact as the ABD. By contrast, the dimensions of the ABD and the apparent average distance between the subdomains do not change significantly during this phase. Because our previous SAXS study did not observe changes in the radius of gyration between 300 μs and 10 ms,22 the slight contraction of the DLD being detected by FRET must be relatively subtle to not significantly affect the overall size of the molecule. The change in FRET efficiency from 0.31 ± 0.01 to 0.41 ± 0.01 can result from an average distance change of only a few angstroms, and it is therefore expected that Rg, which is a root-mean-square average over all interatomic pairwise distances typically measured

338

Microsecond Subdomain Folding in DHFR

Table 4. Root-mean-square end-to-end distances and corresponding FRET efficiencies between the Trp residues and the mutation sites for an unfolded randomcoil polypeptide Acceptor Trp Trp22 Trp30 Trp47 Trp74 Trp133

Arg52 [Å (%)]

Asp87 [Å (%)]

Gln146 [Å (%)]

42 (14) 36 (20) 14 (90) 43 (14) 71 (4.6)

64 (6.0) 59 (7.0) 49 (11) 27 (35) 53 (8.9)

88 (2.7) 85 (2.9) 79 (3.6) 68 (5.1) 27 (35)

The root-mean-square distance was calculated from the simulated distance distribution function of a semiflexible unfolded chain 2 − 3t = 4ð1 − r2 Þ , where r = R/L is the ratio of the model: PðrÞ = 4pNr 9=2 e ð1 − r2 Þ donor–acceptor separation distance R over the contour length L, and t = 3L/2lp, where lp is the persistence length. The constant N is for the normalization of the P(r) distribution. A persistence length of 14 Å and a contour length L of 3.4n were used in the simulations, where n is the number of residues spanning the donor and acceptor residues.36 The root-mean-square end-to-end distances calculated using this model are very close to those obtained using full atom simulations of the unfolded state.37 As a point of reference, it is worth noting that the expected radius of gyration and the hydrodynamic radius for a protein with the size of DHFR are ∼ 40 Å and ∼35 Å, respectively. For an approximate metric for expected random distances in the protein, the most probable distance from a residue near the middle of the chain (e.g., Trp74) to either terminus is ∼ 60 Å, using a semiflexible unfolded chain model.36 The values in parentheses correspond to the average FRET efficiency that takes into account the full distance distribution of the semiflexible chain model and assumes a Förster distance of 22 Å. The FRET efficiency calculations assume an orientation factor corresponding to a full rotational averaging of the donor and the acceptor (κ2 = 2/3). The average FRET efficiencies obtained by considering the full distance distribution are higher than those using a single average distance because the broader distribution in the unfolded state yields a finite probability of sampling shorter distances.

to an accuracy of ± 1 Å, will be insensitive to this change. Formation of two compact subdomains without substantial docking is consistent with a bimodal shape of DHFR over the 300 μs to 10 ms course of the folding reaction.22 In concert with the contraction of the DLD, some portion of Trp residues become buried during the ∼ 550 μs phase (Fig. 3). Since only the DLD showed FRET changes in this time frame, it is likely that Trp residues in the DLD (Trp22, Trp30, and Trp133) are partly sequestered from solvent in this phase. Previous mutational studies showed that DHFR forms a hydrophobic cluster involving Trp30 and Ile155, but not Trp22, within 5 ms of the folding.15,16 A pulsed hydrogen-exchange NMR study showed strong amide protections at Tyr111 (contacting with Trp30) and at Glu157 (close to Trp133 in the native structure). 17 Although these results would be consistent with the notion of Trp30, and possibly Trp133, being buried inside the protein within the τ6 phase, given the ambiguity in assigning FRET pairs

in this study, it is not possible to clarify which Trp residues are buried during the τ6 phase. Folding mechanism of DHFR Our present and previous studies14–19,22,41 provide a more complete picture of the folding mechanism of DHFR (Fig. 6). Unfolded DHFR (Rg ∼ 30 Å) contracts within 35 μs of the initiation of folding to form the I6 intermediate. This intermediate, in which the ABD attains a native-like compactness while the DLD is only partially compacted, is substantially compact (Rg ∼ 23 Å) and heterogeneous. During the τ6 (∼ 550 μs) phase, the DLD further contracts and buries Trp22, Trp30, and/or Trp133. At the end of this kinetic phase, both subdomains achieve the same degree of compactness without a significant change in the overall molecular size. The I5 intermediate formed in the τ6 phase has two subsets of hydrogen-bonding networks corresponding to two hydrophobic clusters, the larger of which is composed mainly of Trp30, Val40, Met92, Ile94, Tyr111, Phe153, and Ile155. During the τ5 (∼200 ms) phase, the structure of the ABD is further organized to form the I1–I4 intermediates; Trp74 is sequestered from solvent, and specific side-chain packing interactions are formed around Val40, Trp47, and Trp74. Finally, during the τ1–τ4 phases, the ensemble of conformers matures to four native-like species that are each capable of binding methotrexate.18 Thus, there emerges a picture of hierarchical assembly in which, after an initial global collapse, the folding of each subdomain evolves independently before docking and establishing global cooperativity. It has previously been suggested that the appearance of the four parallel folding channels reflects alternative subdomain folding or docking modes. Because site-specific pairwise FRET probes would be required, the present data do not provide a test of this hypothesis. Comparison with theoretical predictions By theoretical simulations using simplified Gō models, Clementi et al. predicted that an intermediate with heterogeneous structures—in which the ABD is formed with high probability and the DLD is almost nonexistent—is formed during DHFR folding.42 Using graph-based methods, Zaki et al. predicted that the folding initiation site of DHFR corresponds to residues 40–75 in the ABD, which form a β-α-β motif of the Rossmann fold.43 Although both results are consistent with the structures of the I1–I4 intermediates in that the intermediates have specific side-chain interactions in the ABD, the presence of side-chain packing interactions also in the DLD of the I1–I4 intermediates is contradictory. Rather, these theoretically predicted intermediates are consistent with the I6 intermediate found here in that the

339

Microsecond Subdomain Folding in DHFR

subdomains” model, the subdomain with a continuous sequence structure and low contact order would be the primary determinant of the early stages of folding. By sequestering its sequence components into a compact—perhaps native-like— structure, this subdomain would restrict the search for compact structures by surrounding discontinuous segments in the protein. Thus, the earliest stages in the folding of multi-subdomain proteins might resemble those for small single-domain proteins and might be guided by the same topological principles.22,42 Folding mechanisms of α/β-type proteins The folding mechanism of DHFR is similar to that of the α-subunit of tryptophan synthase, which is composed of a (βα)8 TIM barrel and is also an α/βtype protein.22,33 The folding trajectories of both proteins are intermediary between a type II trajectory (concurrent compaction and secondary structure formation) and a type III trajectory (collapse, followed by secondary structure formation) (see Arai et al.22). Both form an ∼ 30 μs intermediate with heterogeneous structures, although the α-subunit of tryptophan synthase has nonnative interactions that result in an early unproductive kinetic trap. Whether nonnative interactions are present in the I6 intermediate of DHFR is currently unknown, but its swollen structure (Rg ∼ 23 Å) relative to the native state (Rg ∼ 16.6 Å) can allow for the formation of nonnative interactions. Thus, there may be a common folding mechanism for α/β-type proteins, again emphasizing native structure as the primary determinant of the protein folding mechanism.

Materials and Methods Materials

Fig. 6. Cartoon schematic of the folding process of DHFR. Thick and thin black arrows, respectively, show that large and small conformational changes occur in the indicated subdomain during each phase. See the text for details.

Three variants (R52C, D87C, and Q146C) introducing an additional exposed Cys to AS-DHFR27 and five variants (W22L, W30Y, W47L, W74L, and W133Y) replacing a single Trp residue of the wild-type DHFR with Leu or Tyr were constructed by site-directed mutagenesis, as described previously.15,16 Protein expression and purification followed standard protocols, as described previously.16 The extinction coefficients of single Trp substitution mutants were taken from Ohmae et al.28 Urea concentration was measured by a refractive index.44 All experiments were performed in 10 mM potassium phosphate (pH 7.8), 0.2 mM K2EDTA, and 1 mM 2-mercaptoethanol at 23 °C in the presence or in the absence of urea. Protein concentrations in time-resolved fluorescence measurements were 2–6 μM. Protein labeling protocol

ABD is substantially compact but that the DLD is less organized and more extended without specific interactions around Trp30. In this “folding by

IAEDANS (Molecular Probes, Eugene, OR) was labeled according to the procedures described in the protocol

340

Microsecond Subdomain Folding in DHFR

supplied by the manufacturer and by Magg and Schmid, with modifications.45 Before the modification with IAEDANS, 100 μM protein was incubated at room temperature in 10 mM potassium phosphate (pH 7.8), 0.2 mM K2EDTA, 1 mM dithiothreitol, and 4.5 M urea for 45 min to reduce the protein. Then dithiothreitol was removed by passage over a PD-10 column (GE Healthcare) equilibrated with 10 mM potassium phosphate (pH 7.8), 0.2 mM K2EDTA, and 4.5 M urea. The labeling of the mutant protein (∼ 60 μM) was performed in 10 mM potassium phosphate (pH 7.8), 0.2 mM K2EDTA, and 4.5 M urea with a 20-fold excess of IAEDANS. The reaction was performed at room temperature for 12 h in the dark. The labeled protein was purified by two passages over a PD-10 column equilibrated with the buffer for measurement. The iodine atom is removed from IAEDANS after the covalent labeling of AEDANS to the protein. The efficiency of labeling was determined spectrophotometrically using absorbance at 280 nm and 336 nm, as described previously.46 All the labeled proteins showed a stoichiometry of 1:1 AEDANS/protein. Time-resolved Trp fluorescence Details of the TCSPC apparatus equipped with a microsecond CF mixer have been described.23,33 The mixer consisted of 75 μm channels in 127 μm thick PEEK and was held between two fused silica windows by compression. Flow to the microchannel mixer was provided by two syringe pumps (Isco) operating at a combined flow rate of 10 ml min− 1 or 15 ml min− 1, which correspond to dead times of 65 μs and 35 μs, respectively. The dead time was 65 μs for D87AEDANS and Q146AEDANS at 0.45 M urea, and it was 35 μs for all other measurements. Excitation at 293 nm with a repetition rate of 3.8 MHz was provided by the vertically polarized third harmonic of a Ti:sapphire laser. Typical excitation power was ∼0.5–1 mW. Tryptophan emission was measured using a 350 nm interference filter with a 10 nm bandwidth and a 320 nm high-wavelength-pass filter. The variation in excitation intensity along the flow channel was corrected using a standard (N-acetyl-Ltryptophanamide), as described previosuly.23 Separate instrument responses were recorded for each channel by recording of scattered light signal or by numerical deconvolution from the N-acetyl-L-tryptophanamide decay curve. In the equilibrium and CF measurements, the photon counts in the peak channel of a decay curve were typically 30,000 and ∼ 500 counts, respectively. Approximately two to four decay curves were summed at each folding time in the CF experiment. An intensity averaged excited-state lifetime, τave, was calculated as: P 2 ai si i save = P ð1Þ ai si i

where αi and τi are the amplitude and the lifetime of the ith phase in a Trp fluorescence decay curve.32 FRET efficiency was calculated as: EFRET = 1 −

hsiDA hsiD

ð2Þ

where 〈τ〉 is an amplitude averaged lifetime defined as follows:32 P ai si i hsi = P ð3Þ ai i

〈τ〉DA and 〈τ〉D were calculated for IAEDANS-labeled mutants and unlabeled AS-DHFR, respectively. The equilibrium and kinetic data of unlabeled AS-DHFR monitored by 〈τ〉 are shown in Fig. S4, and those of IAEDANS-labeled mutants monitored by τave are shown in Fig. S7. Analyses of time-resolved Trp fluorescence decay curves by the maximum entropy method were performed using an in-house fitting software based on the algorithm described previously.30,47 The equilibrium unfolding transition curves were analyzed assuming a two-state transition between the native state and the unfolded state, using the following equation:     ða1 + a2 cÞ + ða3 + a4 cÞexp − DG0 − mc = RT FðcÞ =     1 + exp − DG0 − mc = RT ð4Þ where ΔG0 is the free-energy change between the native state and the unfolded state in the absence of urea; m is a cooperativity index; c is urea concentration; R and T are the gas constant and the absolute temperature, respectively; a1 and a3 are the y-intercepts of the baselines of the pure native and unfolded states, respectively; and a2 and a4 are the slopes of the baselines of the pure native and unfolded states, respectively.44 The urea concentration at the transition midpoint cM was obtained by ΔG0/m. The difference in ΔG between a mutant and the wild type or AS-DHFR (ΔΔG) was estimated using the following equation:48 DDG =

  1 ðmV + mÞ cM V− cM 2

ð5Þ

where m′ and cM′ are the m and cM of the mutant, respectively. CD and steady-state fluorescence Far-UV CD spectra were measured on an Aviv 62DS spectropolarimeter by scanning from 250 nm to 190 nm at 23 °C. The path length of the cuvette was 1 mm or 2 mm. The protein concentration was 5 μM. The mean residue ellipticity was calculated as described previously.15,16 Fluorescence spectra were measured on an Aviv ATF 105 spectrofluorometer by scanning from 450 nm to 300 nm for unlabeled proteins, and from 570 nm to 300 nm for IAEDANS-labeled proteins at 23 °C (excitation wavelength, 295 nm; bandwidths for excitation and emission, 2 nm each; path length of a cuvette, 10 mm). The protein concentration was 1–2 μM. The equilibrium unfolding transitions by steady-state fluorescence were measured on an Aviv ATF 105 spectrofluorometer from 0 M to 7 M urea at an interval of 0.2 M at 23 °C (excitation wavelength, 292 nm; emission wavelengths, 300–450 nm). The equilibrium unfolding transition curves were analyzed as described previously.16

341

Microsecond Subdomain Folding in DHFR Kinetic folding reactions up to the τ2 phase were measured on an SX.18MV-R stopped-flow spectrometer (Applied Photophysics Ltd., UK) (path length, 2 mm; dead time, 6 ms; excitation wavelength, 292 nm). The emission wavelengths were above 320 nm and 395 nm, with cutoff filters provided by the manufacturers for the unlabeled and IAEDANS-labeled proteins, respectively. The protein concentrations were 2 μM. The time-dependent change in fluorescence intensity was fit by the method of nonlinear least squares, as described previously.16 The τ1 and τ2 phases were measured by the manual-mixing method on an Aviv ATF 105 spectrofluorometer (excitation wavelength, 292 nm). The emission wavelength was 335 nm and 500 nm for the unlabeled and IAEDANS-labeled proteins, respectively.

9.

10. 11. 12.

13.

Acknowledgements We thank Aiko Fujisawa, Meiko Oose, Gou Sarara, and Tatsuyuki Takenawa for protein purification. This work was supported, in part, by Grantsin-Aids for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan to M.A. and by grant MCB0327504 (National Science Foundation) from the US National Science Foundation (C.R.M. and O.B.).

14.

15.

16.

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2011.04.057

17.

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