Mapping the Transition State for DNA Bending by IHF

doi:10.1016/j.jmb.2012.02.028

J. Mol. Biol. (2012) 418, 300–315 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

Mapping the Transition State for DNA Bending by IHF Paula Vivas 1 †‡, Yogambigai Velmurugu 1 †, Serguei V. Kuznetsov 1 , Phoebe A. Rice 2 and Anjum Ansari 1, 3 ⁎ 1

Department of Physics (M/C 273), University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607, USA Department of Biochemistry and Molecular Biology, University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA 3 Department of Bioengineering (M/C 063), University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607, USA 2

Received 14 March 2011; received in revised form 14 February 2012; accepted 17 February 2012 Available online 24 February 2012 Edited by D. E. Draper Keywords: protein–DNA interactions; laser temperature jump; DNA bending dynamics; FRET measurements; integration host factor

How DNA-bending proteins recognize their specific sites on DNA remains elusive, particularly for proteins that use indirect readout, which relies on sequence-dependent variations in DNA flexibility/bendability. The question remains as to whether the protein bends the DNA (protein-induced bending) or, alternatively, “prebent” DNA conformations are thermally accessible, which the protein captures to form the specific complex (conformational capture). To distinguish between these mechanisms requires characterization of reaction intermediates and, in particular, snapshots of the transition state along the recognition pathway. We present such a snapshot, from measurements of DNA bending dynamics in complex with Escherichia coli integration host factor (IHF), an architectural protein that bends specific sites on λ-DNA in a U-turn by creating two sharp kinks in DNA. Fluorescence resonance energy transfer measurements in response to laser temperature-jump perturbation monitor DNA bending. We find that nicks or mismatches that enhance DNA flexibility at the site of the kinks show 3- to 4-fold increase in DNA bending rates that reflect a 4- to 11-fold increase in binding affinities, while sequence modifications away from the kink sites, as well as mutations in IHF designed to destabilize the complex, have negligible effect on DNA bending rates despite N250-fold decrease in binding affinities. These results support the scenario that the bottleneck in the recognition step for IHF is spontaneous kinking of cognate DNA to adopt a partially prebent conformation and point to conformational capture as the underlying mechanism of initial recognition, with additional proteininduced bending occurring after the transition state. © 2012 Elsevier Ltd. All rights reserved.

Introduction *Corresponding author. E-mail address: [email protected]. † P.V. and Y.V. contributed equally to this work. ‡ Present address: P. Vivas, Physics Department, Ohio State University, Columbus, OH 43210, USA. Abbreviations used: T-jump, temperature jump; IHF, integration host factor; FRET, fluorescence resonance energy transfer; wt, wild type; CAP, catabolite-activator protein; EDTA, ethylenediaminetetraacetic acid.

Many cellular processes involve interactions between proteins and DNA in which proteins recognize and bind tightly to specific sites on the DNA with thousand- or million-fold higher affinities than to random DNA sequences and that kink, bend, or twist DNA at that site. 1–8 A large number of these DNA-bending proteins find their preferred sites by “indirect readout” of the DNA sequence, which relies on the sequence dependence of the DNA's

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Transition State for DNA Bending by IHF

conformation and deformability rather than direct interactions between the protein and specific DNA base pairs. 9 The protein also adjusts its conformation to facilitate favorable interactions. 10 These concerted rearrangements in protein and DNA are fundamental to the underlying recognition mechanism. However, a detailed understanding of the mechanism is lacking. In particular, a key question remains: does the protein bend the DNA (proteininduced bending) or is the bent conformation thermally accessible to DNA in the absence of the protein, which the protein then readily captures to form a tight complex (conformational capture). The interplay between conformational capture and induced fit in bimolecular recognition has garnered a lot of attention. 11–18 To distinguish between the two mechanisms requires measurements of the kinetics of conformational rearrangements that lead to the formation of the final complex, and, in particular, snapshots of the transition-state ensembles or bottlenecks along the recognition pathway. The recognition pathways of a few protein–DNA complexes have been characterized in detail, including that of the TATA-box binding protein 19–21 and of the E2 protein from human papillomavirus (HPV E2). 22–24 For these and other DNA-bending proteins, there is ample evidence that DNA sequences that are more easily deformed into the conformation adopted by the DNA in the complex bind to the protein with a higher affinity. 25–33 The question remains: is the enhanced affinity reflected in an increase in the DNA bending rate or a decrease in the unbending/dissociation rate? Is the DNA already bent in the transition-state ensemble that separates the nonspecific from the specific complex? Previous kinetics measurements on DNA-bending proteins have primarily used stopped-flow techniques that yielded valuable information on the overall association and dissociation kinetics but were unable to capture the kinetics of DNA bending. 34–39 Thus, for most DNA-bending proteins, even the simplest question remains unanswered: on what timescale does the DNA bend in the complex? One notable exception is integration host factor (IHF), an architectural protein from the IHF/HU family of DNA-bending proteins that are ubiquitous in eubacteria and that aid in chromosomal compaction as well as in the assembly of higher-order nucleoprotein complexes necessary for replication initiation, some site-specific recombination reactions, and transcriptional regulation of certain genes. 40–46 This family of proteins interacts exclusively with the minor groove and backbone of the DNA and provides a paradigm for investigating the indirect readout mechanism for recognition of DNA binding sites as well as the mechanics of DNA bending. 47 IHF is a 20-kDa heterodimer of related subunits that recognizes asymmetric DNA binding sites. In

301 the crystal structure of IHF bound to one of its cognate sites (the H′ site of phage λ), the 35-bp DNA segment is bent by almost 180°. 3 Most of the DNA distortion occurs at two sharp “kinks” spaced 9 bp apart (Fig. 1). IHF has two long β-ribbon “arms” that wrap around the DNA, and the kinks in the DNA are stabilized by the intercalation of highly conserved proline residues located in these arms. The final complex is stabilized by additional, primarily electrostatic, interactions, with the DNA wrapped around a positively charged surface of IHF. In vivo, this bending allows distal regions of DNA to come together so that other proteins bound to them can interact. 46 Kinetics of DNA bending in the IHF–H′ complex were revealed in stopped-flow and laser temperature-jump (T-jump) studies, using fluorescence resonance energy transfer (FRET) measurements on end-labeled DNA substrates, 47–50 which showed DNA bending on timescales of 1–10 ms, very similar to the timescales for thermal disruption of a single A-T base pair in B-DNA. 51,52 These results suggested that local distortions of the DNA structure, as a consequence of thermal fluctuations in base pairing nucleated at regions of weak stacking, may be sufficient to overcome the free-energy barrier needed to partially bend/kink DNA prior to forming a tight complex with IHF. 49 The DNA bending rates were found to be independent of the salt concentration, 50 indicating that any conformational rearrangements in the complex that are accompanied by ion release occur after the ratelimiting step. These studies pointed to a picture of the transition-state ensemble as one in which the DNA is partially bent/kinked, but stabilizing interactions between IHF and DNA have not yet been made. Here, we report measurements that directly probe the nature of the transition-state ensemble and examine the role of sequence-dependent DNA flexibility in the ability of IHF to recognize its cognate site. We investigated changes in the DNA bending dynamics on modified DNA substrates with nicks or mismatches at the kink sites designed to enhance local DNA flexibility. We also investigated the effect of sequence modifications in the recognition site removed from the kinked regions and the effect of mutant IHF proteins designed to destabilize the complex by perturbing the electrostatic interactions. We wished to determine to what extent the changes in the binding affinity, as a result of these modifications, were reflected in the bending rates. Modifications that make the DNA more flexible/bendable at the site of the kink are expected to accelerate the DNA bending rates. In contrast, if our hypothesis is correct, in that specific protein– DNA interactions that stabilize the final complex are made after the system passes through the transitionstate ensemble, then we expect to see no change in

302

Transition State for DNA Bending by IHF

Fig. 1. Cocrystal structures of the IHF/HU family of DNA-bending proteins. (a) IHF–DNA cocrystal structure [Protein Data Bank (PDB) code: 1IHF]. The α- and β-chains of the IHF protein are shown in blue and green, respectively, with the conserved proline residues indicated in red. The DNA is shown in gray, with locations of modifications in the DNA sequences used in this study highlighted in color: yellow sphere, site of a nick in the sugar-phosphate backbone in the NickC substrate; pink, mismatches introduced in the TT8AT, TT-loop, and AT-loop substrates; red, T → A mutation in the H′44A substrate. (b) Sequence of the DNA substrate in the cocrystal structure with IHF, which contains the H′ binding site from bacteriophage λ. The DNA was nicked at the position indicated by the black arrow, to facilitate crystal packing. In the complex, the DNA is sharply kinked at the two sites indicated by the blue arrows. (c) Side view of solventaccessible surface representation of IHF. Regions of positive and negative electrostatic potential are shown in blue and red, respectively. (d) The ensemble of 25 NMR structures of the homodimeric HU protein from Bacillus stearothermophilus (PDB code: 1HUE) is shown in gray. One of these structures is highlighted, in blue and green with conserved proline residues in red. (e) The structure of the IHF–H′ complex (1IHF), rotated by 90° relative to the view in (a). The β-ribbon arms of the proteins of this family are more flexible in the absence of a bound DNA. Upon binding, they wrap around the DNA and lie in the minor groove, and the prolines intercalate into the kinks in the DNA.

the DNA bending rates as a result of destabilizing modifications that are located away from the kink sites. The spirit of these measurements is analogous to the φ-value analysis pioneered by Fersht to probe the transition-state ensemble for protein folding. 53 Although similar approaches have been used to examine the encounter complex in stopped-flow studies of protein–DNA interactions, 23,24,54–56 this study provides the first snapshot of the transitionstate ensemble separating the nonspecific encounter complex and the final, fully bent complex for a DNA-bending protein.

Results Overview We carried out measurements on the IHF–H′ complex and on several variants of IHF and H′, to examine the effect of changes in complex stability on DNA bending rates. Variants of the H′ substrate were designed to enhance the flexibility/bendability of DNA by introducing a nick in the sugar-

phosphate backbone (NickC) or mismatches (TT8AT, AT-loop, TT-loop) at or near the kink sites (Fig. 2). Another variant (H′44A) was chosen to perturb interactions distal to the kinks. 57,58 Position 44 in the DNA (using phage λ numbering) is in the center of the TTR consensus region and located 6 bp from one of the kinks (Fig. 1a). A T → A mutation at that site is known to significantly destabilize the complex 58 but without disturbing the bendability of DNA at the kink sites. We also examined the effects of mutations in the protein (αK5A, βK84A, and αR21C), in which positively charged residues at different sites on the DNA-contacting surface of the protein, as indicated in Fig. 8a, were mutated to neutral ones. These mutations are expected to destabilize the complex by disrupting ionic interactions at different positions along the IHF– DNA interface. Here, we describe equilibrium and kinetics measurements on each of these complexes that allow us to determine to what extent changes in binding affinity are reflected in the bending rates, thus providing insights into the accessible conformations and interactions within the transition-state ensemble.

303

Transition State for DNA Bending by IHF

Fig. 2. Sequences of H′ substrate and its variants. The intact H′ substrate is identical with the sequence shown in Fig. 1b and contains no nicks. The 5′-end of the top strand is fluorescein-dT (yellow), and the 5′-end of the bottom (complementary) strand is TAMRA-dT (dark pink). The consensus region is shown in gray. The blue arrows indicate locations of kinks in the DNA when in complex with IHF. In NickC, the sugar-phosphate backbone of the bottom strand is nicked at the location indicated by the black arrow. In H′44A, modification of a base pair in the TTR consensus region is shown in red. In TT8AT, TT-loop, and AT-loop, mismatches inserted at or near the site of the kinks are shown in pink. The locations of these modifications are also indicated in Fig. 1a.

Dissociation constants for IHF–H′ and its variants measured with equilibrium FRET The ∼ 180° bending of the DNA in the IHF–H′ cocrystal structure (Fig. 1a) shortens the DNA endto-end distance from ∼ 100 Å to ∼50 Å. 3 Therefore, FRET between fluorophore-labeled DNA ends provides a sensitive probe for direct measurement of DNA bending in complex with the protein. 48,49,59 We labeled the 5′-termini of one DNA strand with fluorescein (F) and the other with TAMRA (R) and used equilibrium FRET measurements to monitor the association/dissociation equilibrium as well as relaxation kinetics in response to laser T-jump. IHF binds tightly to the H′ substrate, with dissociation constant Kd ≈ 25 pM in 100 mM KCl at 20 °C, as obtained from ratio of association and dissociation rates measured directly in stopped flow. 48,60 For such high-affinity complexes, equilibrium titration measurements that require at least nanomolar protein and DNA concentrations yield highly inaccurate Kd values. 50 In order to obtain

reliable Kd estimates in the sub-nanomolar range from equilibrium FRET measurements, we use an alternative approach whereby we measure the FRET efficiency of the complex at a fixed protein–DNA concentration but for varying salt concentrations, to disrupt the complex, and parameterize the salt dependence of the dissociation constant as Kd ∼ [salt] SK d (see Supplementary Text for details). The Kd values determined using this “salt titration” approach at three different KCl concentrations (100, 200, and 300 mM) together with the values of the slope parameter SKd are summarized in Table 1 for each of the complexes. We are primarily interested in a quantitative comparison of Kd for different complexes at 100 mM KCl, at which the bulk of the kinetics measurements was done. In all cases, with the exception of IHF– NickC, the difference in binding affinities between the variants and the wild-type (wt) IHF–H′ complex, determined using the salt titration approach, is larger than the uncertainty in the

Table 1. Dissociation constants and relaxation rates for IHF–DNA complexes

IHF–H′ IHF–NickC IHF–TT8AT IHF–H′44A αK5A–H′ βK84A–H′ αR21C–H′ a

Kd (in M)a (0.1 M KCl)

Kd (in M)a (0.2 M KCl)

Kd (in M)a (0.3 M KCl)

SKDa

kr (s− 1) at 25 °C

kr (s− 1) at 37 °C

2.7 ± 0.2 × 10− 11 (2.5 × 10− 11) b 2.5 ± 1.8 × 10− 11 (7.0 × 10− 12)b 2.5 ± 1.2 × 10− 12 7.4 ± 2.8 × 10− 9 3.5 ± 3.0 × 10− 8 1.6 ± 0.3 × 10− 8 1.0 ± 0.2 × 10− 11

4.6 ± 0.4 × 10− 8 2.0 ± 1.3 × 10− 8 2.0 ± 0.7 × 10− 9 5.8 ± 3.3 × 10− 6 1.5 ± 0.5 × 10− 6 1.1 ± 0.1 × 10− 6 2.0 ± 0.1 × 10− 8

3.0 ± 2.0 × 10− 6 9.2 ± 0.9 × 10− 7 1.3 ± 0.2 × 10− 7 4.3 ± 3.3 × 10− 4 1.3 ± 0.1 × 10− 5 1.3 ± 0.2 × 10− 5 1.8 ± 0.2 × 10− 6

10.3 ± 1.0 9.6 ± 0.5 10.1 ± 0.3 10.0 ± 1.1 5.4 ± 0.5 6.1 ± 0.3 11.0 ± 0.3

149 ± 30 377 ± 60 547 ± 84 177 ± 38 211 ± 21 180 ± 36 102 ± 10

475 ± 95 1075 ± 170 1904 ± 292 459 ± 98 637 ± 64 385 ± 78 329 ± 30

kr/krref Kdref/Kd (0.1 M KCl) (0.1 M KCl) 1 1.1 (3.6)b 10.8 0.004 0.0008 0.002 2.7

1 (1)c 2.5 (2.3) 3.7 (4) 1.2 (1) 1.4 (1.3) 1.2 (0.8) 0.7 (0.7)

All Kd values are obtained in 25 °C in buffer: 20 mM Tris–Cl (pH 8.0), 100 mM KCl, 1 mM EDTA, and 0.01% NP-40. Values in parenthesis are from ratio of association and dissociation rates measured in stopped flow at 20 °C.48 c The values are at 25 °C, obtained from extrapolation of the Arrhenius fit to the measured relaxation rates versus inverse temperature; the values in parenthesis are interpolated at 37 °C. b

304 measurements. However, for the IHF–NickC complex, the uncertainties in the Kd estimates are larger than the difference in Kd between IHF– NickC and IHF–H′, measured previously with stopped flow under identical buffer condition by the Crothers group 48,60 (see Table 1). Therefore, to quantitatively interpret our kinetics results on IHF–NickC, we use instead the more accurate Kd values obtained from stopped flow. The results from the equilibrium measurements can be summed up as follows: (i) 4-fold increase in affinity (decrease in Kd) for IHF–NickC, (ii) 11-fold increase in affinity for IHF–TT8AT, (iii) 250-fold decrease in affinity for IHF–H′44A, (iv) 500- to 1200fold decrease in affinity for αK5A–H′ and βK84A–H′, and (v) b 3-fold change in affinity for αR21C–H′. Our results on substrates with nicks (NickC) and mismatches (TT8AT) are consistent with previous studies that showed enhanced affinities of IHF for substrates that are rendered more flexible at the site of the kinks. 30,60,61 In contrast, modifications in the consensus region of H′ (as in H′44A) or mutations in IHF that reduce the positive charge on the wrapping surface of the protein significantly destabilized the complex, as anticipated. 58 One of the mutants (αR21C) had a negligible effect on the binding affinity for H′ presumably because the 35-mer-long H′ substrate is not long enough to make stabilizing contacts with the Arg in this position in the wt protein (Fig. 8a), although this residue appears to play a role in stabilizing the crystalline form by making contacts with DNA segments from the neighboring complexes. Thermal stability of IHF Prior to carrying out the T-jump measurements, we examined the stability of the IHF–H′ complex against thermal denaturation. The thermal unfolding of IHF was monitored using circular dichroism (CD) as well as by fluorescence of intrinsic Tyr residues, while the stability of H′ was monitored using absorbance changes at 266 nm (Supplementary Fig. S1). No changes in the CD spectra are observed for IHF protein below 40 °C, with a thermal unfolding transition observed at 62 °C from CD measurements at 222 nm, 58 °C from CD measurements at 276 nm, and 57 °C from fluorescence emission of Tyr measured at 304 nm. For the IHF–H′ complex at 5 μM:5 μM concentrations, the melting transition measured with Tyr fluorescence is shifted to higher temperatures, with the onset of the transition occurring near 60 °C, and the midpoint of the transition shifted to 70 °C. Interestingly, the thermal denaturation profile obtained for the IHF–H′ complex is very close to the duplex melting profile of H′ by itself, with a melting temperature of 68 °C at a strand concentration of 4.5 μM. Thus, in the absence of bimolecular

Transition State for DNA Bending by IHF

Fig. 3. Bimolecular kinetic scheme for binding of IHF to DNA. The bimolecular association/dissociation step is indicated by kns and k− ns, and the unimolecular DNA bending/unbending step is indicated by kbend and kunbend.

disruption, the IHF–H′ complex is stable up to nearly 60 °C in 100 mM KCl. Time-resolved FRET measurements: Unimolecular bending/unbending versus bimolecular association/dissociation We monitored relaxation kinetics of IHF–DNA complexes in response to the laser T-jump perturbation. Briefly, we used an IR laser pulse to heat the sample by ∼ 5–10 °C and then monitored the increase in donor intensity resulting from decreasing FRET efficiency between the ends as the complex re-equilibrated to a conformation where the DNA was less bent. In the minimal kinetic scheme describing protein–DNA interactions (Fig. 3), the relaxation kinetics after a T-jump perturbation are expected to have contributions from (i) unimolecular bending/unbending of the DNA within the complex and (ii) bimolecular disruption of the complex, as was observed in T-jump measurements on the binding and wrapping of single-stranded DNA around a single-strand binding protein, 62 and for the IHF–H′ complex under high salt (N 200 mM KCl) conditions. 50 Previously, we demonstrated that, for the IHF–H′ complex, the decrease in FRET with increasing temperature is independent of [IHF] and [DNA] in the concentration range of 200 nM to 5 μM in 100 mM KCl, at least up to ∼40 °C (Fig. 4a), and in the concentration range of 1 μM to 18 μM in 200 mM KCl, up to ∼ 60 °C (Supplementary Fig. S5). 49,50 Thus, for [KCl] ≤ 200 mM and IHF–H′ concentrations ≥ 1 μM each, all of the observed change in FRET up to 60 °C is from unimolecular unbending of the DNA in the complex, with no significant evidence of bimolecular disruption. All T-jump measurements reported here were carried out at IHF–H′ concentrations in the range of 5– 10 μM each, and the observed relaxation kinetics reflect unimolecular bending/unbending of the complex. For most variants of the IHF–H′ complex, it was verified from the concentration dependence of the equilibrium FRET measurements that relaxation kinetics under experimental conditions for the Tjump measurements reflected only unimolecular bending/unbending rather than bimolecular disruption and rebinding. For these variants,

Transition State for DNA Bending by IHF

305 DNA bending rates in the IHF–H′ complex are similar to the rates at which a single base pair opens transiently in B-DNA

Fig. 4. Equilibrium and kinetics measurements on IHF– H′. (a) FRET efficiency (E) as a function of temperature is plotted for the IHF–H′ complex at three different concentrations of IHF and H′: ( ) (5 μM:5 μM), ( ) (1 μM:1 μM) and ( ) (200 nM:200 nM). (b) The maximum of the donor fluorescence emission spectra of the IHF–H′ complex (I complex ) (●) is plotted as a function of temperature. The red line is the corresponding intensity of the H′ substrate in the absence of IHF (Ifree) and represents the temperature dependence of the donor quantum yield. The two measurements have been normalized to match at 10 °C. (c) Fluorescence emission intensity for the IHF–H′ complex, in response to an ∼ 5 °C T-jump (from 40 °C to 45 °C), is plotted as a function of time. Negative values are assigned to the time points prior to the infrared laser pulse that induces a T-jump. The continuous line represents a fit to the data as described in Supplementary Text. (d) Control experiment on donoronly strand of H′; the continuous line is a singleexponential fit with a time constant of ∼ 200 ms and reflects the relaxation of the sample temperature to the pre-jump value.

unimolecular relaxation kinetics could be measured directly at the same ionic strength (100 mM KCl). The one exception was IHF–NickC, which required careful extrapolation from a series of measurements at higher salt (see below).

A typical relaxation trace for the IHF–H′ complex, monitored by measuring changes in the donor fluorescence in response to a T-jump perturbation, is shown in Fig. 4c. The quantum yield of the donor initially drops as a result of the change in temperature after the laser pulse and then rises as the protein–DNA complexes relax to a lower-FRET state reflecting a new equilibrium at the higher temperature (Fig. 4b). Control experiments on donor-only labeled strand of H′ show only the initial drop in quantum yield and then a slow relaxation characteristic of the dissipation of heat from the heated volume of the sample and the recovery of the temperature to that of the surrounding bath (Fig. 4d). Relaxation kinetics traces exhibit deviations from single-exponential behavior, even for the unimolecular bending/unbending process, indicating that additional intermediates may be needed in the minimal kinetic scheme of Fig. 3. For the purpose of this study, we report a single average relaxation rate (see Supplementary Text for details). The relaxation rates, obtained from a series of measurements at different initial (before T-jump) and final (after T-jump) temperatures, are plotted versus the final temperature in Fig. 5. Both kbend and kunbend (Fig. 3) formally contribute to the observed relaxation rates. However, a global analysis of the relaxation rates and binding affinities under varying salt conditions, in terms of the microscopic rate constants shown in the scheme of Fig. 3, revealed that, under the conditions of our measurements

Fig. 5. Relaxation rates measured for IHF–H′. The relaxation rates, kr, for the IHF–H′ complex are plotted versus inverse (final) temperature (Tf) as obtained from three sets of T-jump measurements (●,■,○) under conditions where the T-jump perturbation does not dissociate the IHF–H′ complex. The plateau values of the stopped-flow relaxation rates at high [IHF] are also shown (▲; data from Sugimura and Crothers 48). The shaded areas represent the range of base-pair opening rates from imino proton exchange measurements, from Coman and Russu 52 (pink, A:T; blue, C:G). The red vertical bar is the A:T base-pair opening rate for the H′ sequence, at the site of one of the kinks, from Dhavan et al. 51

306

Transition State for DNA Bending by IHF

Fig. 6. Kinetics measurements on IHF–NickC for varying [KCl]. (a) Relaxation rates kr obtained from T-jump measurements are plotted versus inverse temperature for varying concentrations of KCl: ( ) 220 mM, ( ) 300 mM, ( ) 397 mM, ( ) 450 mM and ( ) 500 mM. (b) The relaxation rate kr at 37 °C is plotted versus [KCl]. The kr values at this temperature were obtained from interpolation (or extrapolation) of the data in (a). The continuous line is a phenomenological fit to the salt dependence, assuming a plateau at low salt, and the open circle is the value of kr extrapolated to 100 mM KCl. (c) Relaxation rates kr for the IHF–NickC complex, obtained at 100 mM KCl, are plotted as a function of inverse temperature; ( ) data points are from extrapolation from T-jump measurements carried out at higher ionic strength, as shown in (b); ( ) data points are the unimolecular rates obtained from stopped-flow measurements (from Sugimura 60). The continuous line is an Arrhenius fit to the IHF–NickC data; the broken line is the corresponding Arrhenius fit for the IHF–H′ data, reproduced from Fig. 5.

(100 mM KCl), kbend ≫ kunbend, and thus, the primary contribution to the observed kr is the DNA bending rate kbend. 63 The relaxation rates obtained from the T-jump measurements are in excellent agreement with the unimolecular bending rates obtained from stopped-flow measurements of Sugimura and Crothers 48 (Fig. 5) and are well described in terms of an Arrhenius temperature dependence in the ∼ 10–60 °C range, with an apparent activation enthalpy of 18 ± 3 kcal/mol. The data in Fig. 5 show that the DNA bending rates in the IHF–H′ complex are similar to the rates at which a single base pair opens transiently in BDNA, as obtained from NMR measurements of imino proton exchange. 51,52 In contrast to C:G base pairs, A:T base pairs show a wide range of opening times that depend on the sequence context, 52 presumably due to the sequence dependence of the stacking interactions that must also be disrupted and which may present the dominant barrier to A:T base-pair disruption. The cocrystal structure of IHF– H′ and structures of other members of this family (HU and Hbb) bound to their cognate sites show complete disruption of base stacking at the kinks, 3,64,65 but no disruption of the Watson–Crick pairs. We therefore suggest that spontaneous thermal disruption in base stacking, nucleated at regions of weak stacks, contributes to the ratelimiting step in the kinetics of DNA bending in complex with IHF. A nick at a kink site increases DNA bending rates The Crothers group had previously shown that nicks in the H′ substrates at or near the site of the kinks accelerate the DNA bending rates. 48,60 In

some cases, the bending rates became too fast to be completely resolved on the stopped-flow timescales. The time resolution afforded by laser T-jump allows us to better resolve the unimolecular bending step over a wide temperature range. We started with equilibrium and kinetics measurements on the IHF–NickC complex under ionic conditions of 100 mM KCl, identical with that used by the Crothers group. However, unlike in the IHF–H′ complex, we were either unable to perturb the complex, at temperatures below 40 °C, or we completely disrupted the complex, with apparently very slow bimolecular association/dissociation kinetics that were not observed on our T-jump time window (Supplementary Fig. S6). We next increased [KCl] to more easily perturb the IHF– NickC complex and recovered the relaxation kinetics within the T-jump time window, for [KCl] in the range of 220–500 mM (see Supplementary Text). The relaxation rates for the IHF–NickC complex obtained at different temperatures and different [KCl] concentrations are summarized in Fig. 6a. From these data, we obtained the [KCl] dependence of the relaxation rates at a given temperature (as shown in Fig. 6b for data at 37 °C). The measurements reveal a nonlinear dependence of log(kr) versus log([KCl]), similar to our observation on the IHF–H′ complex (Supplementary Fig. S5c). As demonstrated in our earlier study, 50 this nonlinear dependence arises because, at low [KCl], the observed relaxation kinetics on the T-jump time window are from the unimolecular process, and the corresponding relaxation rate is independent of [KCl], while at high [KCl], the observed relaxation kinetics include contributions from the bimolecular association/

307

Transition State for DNA Bending by IHF

Fig. 7. Equilibrium and kinetics measurements on IHF–TT8AT and IHF–H′44A. (a) FRET efficiency versus temperature at four different concentrations of IHF–TT8AT: ( ) 20 μM:20 μM, ( ) 12.5 μM:10 μM, ( ) 5 μM:5 μM and ( ) 1 μM:1 μM. All FRET efficiency values have been normalized to 1 at the lowest temperature (∼20 °C). (Inset) The actual FRET values for each sample at 20 °C are plotted as a function of the IHF concentration in that sample. (b and c) Relaxation rates kr obtained from T-jump measurements are plotted as a function of the inverse temperature for (b) IHF–TT8AT ( ) and IHF–H′44A ( ), and (c) IHF–AT-loop ( ) and IHF–TT-loop ( ). For each sample, the relaxation rates obtained from two sets of measurements, at IHF–DNA concentrations of 12.5 μM:10 μM and 20 μM:20 μM, are averaged together. The error bars are calculated from the standard deviations of all the measured relaxation rates from an Arrhenius fit to the data, shown as the continuous lines. The broken line in (b) and (c) is the Arrhenius fit to the IHF–H′ data, reproduced from Fig. 5.

◆

dissociation process, and the corresponding relaxation rate decreases with increasing [KCl]. To obtain the unimolecular relaxation rates for the IHF–NickC complex at 100 mM KCl, we obtained the plateau value in the limit of low [salt] (as shown in Fig. 6b) at several different temperatures (Fig. 6c). The relaxation rates thus obtained are in excellent agreement with the unimolecular rates obtained from the stopped-flow measurements on the IHF– NickC complex (Fig. 6c), thus providing a reassuring confirmation that our extrapolation from N 100mM KCl measurements is sufficiently accurate. The combined temperature dependence yields an apparent activation enthalpy of 16 ± 3 kcal/mol. These rates, which we identify as the DNA bending rates, are 377 s − 1 for the IHF–NickC complex at 25 °C in comparison with 149 s − 1 for the IHF–H′ complex. Thus, DNA bending in IHF–NickC in 100 mM KCl is 2.5-fold faster than in IHF–H′, an enhancement that is similar to the 3.6-fold increase in the binding affinity of IHF for NickC in comparison with intact H′ and which indicates that a significant fraction of the change in the binding affinity appears in a corresponding change in the bending rates. Mismatches at the kink sites enhance DNA bending rates We next carried out kinetics measurements on modified H′ substrates with mismatches (internal loops) located at or near the sites of the kinks. The choice of these modifications was motivated by studies carried out by Grove et al., who demonstrated that mismatches introduced in the H′ sequence at or near the site of the kinks (∼ 8–9 bp apart) increased the binding affinity of IHF for the H′

substrate. 30 For the TT8AT variant (Fig. 2), which is composed of two 4-nucleotide loops located at or near either kink, Grove et al. observed a 15-fold increase in binding affinity. Under our buffer conditions, we measure 11-fold increase in binding affinity for IHF–TT8AT in comparison with IHF–H′ (Table 1). Equilibrium FRET measurements on the IHF– TT8AT complex as a function of temperature showed that the change in FRET with increasing temperature is independent of the IHF and DNA concentrations in the range of 20–40 °C but starts to diverge above ∼ 40 °C, especially at IHF–DNA concentrations below 5 μM (Fig. 7a). At higher IHF–DNA concentrations (10 μM or greater), the change in FRET remains independent of the concentration up to nearly 50 °C. These measurements indicate that, at concentrations used in the T-jump measurements (see below), the change in FRET is primarily from unbending within the complex. Kinetics measurements on the IHF–TT8AT complex, carried out at two different IHF–DNA concentrations (12.5 μM:10 μM and 20 μM:20 μM) showed no systematic dependence of the observed relaxation rates on the concentrations, in the temperature range 26–52 °C, reaffirming that the observed kinetics are primarily unimolecular. The relaxation rate averaged over the two sets of measurements is 547 s − 1 for the IHF–TT8AT complex at 25 °C, which is ∼ 4-fold faster than that for the IHF–H′ complex (Fig. 7b and Table 1). Therefore, as in the case of the nicked substrate, mismatches at the kink sites also accelerate DNA bending rates, although in this case, the enhanced DNA bending rates do not fully account for the 11-fold increase in binding affinity of IHF for TT8AT.

308

Transition State for DNA Bending by IHF

Fig. 8. Equilibrium and kinetics measurements on mutant IHF–H′. (a) The IHF–H′ cocrystal structure (PDB code: 1IHF) is shown with location of three positively charged residues, shown in red, that were substituted for a neutral residue: (top) βK84A, (middle) αK5A, and (bottom) αR21C. (b) FRET efficiency versus temperature for the αK5A–H′ complex ( , ) and the βK84A–H′ complex ( , ), obtained at two different protein–DNA concentrations: 20 μM:20 μM (open circles) and 1 μM:1 μM (filled circles). The data are normalized to match at the lowest temperature. The insets show the actual FRET values for each sample at 20 °C; the broken line represents the average FRET value for the IHF–H′ complex. (c) The relaxation rates kr, obtained from T-jump measurements on αK5A–H′ ( ), βK84A–H′ ( ) and αR21C–H′ ( ), are plotted versus inverse temperature. The continuous lines are Arrhenius fits to the relaxation rates for each of the mutants; the broken line is the corresponding fit for the IHF–H′ complex, reproduced from Fig. 5.

To investigate the contribution to the enhanced bending rates from each kink site, we also carried out kinetics measurements on (i) the AT-loop, with the mismatch on one side, and (ii) TT-loop, with the mismatch on the other side (Fig. 2). The relaxation rates for the IHF–AT-loop and IHF–TT-loop complexes are 357 s − 1 and 665 s − 1, respectively, at 25 °C, which is two to four times faster than that for IHF–H′ (Fig. 7c). Thus, the enhancement in the bending rates that we observe in the TT8AT substrate cannot be attributed solely to one kink or the other and reflects enhanced flexibility of the entire substrate. A DNA mutation distal to the kink sites decreases affinity but does not affect bending rates In the H′44A sequence, a single T → A mutation in the TTR consensus region of the H′ sequence results in a 250-fold decrease in the binding affinity in comparison with H′ (Table 1). Previous measurements, using gel mobility shift assays, had indicated ∼ 100-fold decrease in the binding affinity for H′44A relative to H′. 58 The FRET efficiency for the IHF–H′ 44A complex is ∼ 0.45 at 25 °C, significantly smaller than the ∼0.55 FRET efficiency obtained for the IHF–H′ complex under identical solvent conditions. This result indicates that, despite the very similar crystal structures of IHF–H′ and IHF–H′44A complexes, 58 the average conformation of H′44A in the complex is less bent under solution conditions, consistent with weaker interactions between IHF and one of the flanking “arms” of the bent DNA. Crystallographic studies show that a very high twist in the TG step of the TTG trinucleotide in the original H′ consensus sequence allows formation of

a chain of salt bridges (ionic interactions) involving three amino acid residues of IHF and the DNA itself. 58 In the H′44A sequence, the AG step of the TAG sequence is less flexible, and in complex with IHF, the twist is spread out more evenly among the dinucleotide steps, thus disrupting the specific ionic interactions, but without disturbing the bendability of DNA at the kink sites. Our prediction was that the DNA bending rates observed in the IHF–H′44A complex should remain unchanged in comparison with the IHF–H′ complex, despite the significant decrease in stability. Kinetics measurements confirm this prediction (Fig. 7b), indicating that all changes in the free energy of the complex, as a result of these modifications, are reflected in the unbending/ dissociation rates, and not in the bending rates. We conclude that the transition state separating the specific from the nonspecific complex is unperturbed as a result of the H′44A modification, that is, specific interactions that IHF makes with the TTR consensus site of H′ are not yet made in the transition-state ensemble. Protein mutations distal to the kink sites also affect affinity with negligible effect on bending rates We next examined the effect of IHF mutants, designed to perturb the complex, on the DNA bending rates. Three IHF mutants were studied, in which positively charged residues (Lys or Arg) at increasing distances from the kink sites were replaced with neutral residues (Fig. 8a). For the three mutants that were investigated in this study, αK5A–H′, βK84A–H′, and αR21C–H′, the dissociation constants Kd were determined to be 35 nM, 16 nM, and 10 pM, respectively, in comparison with

Transition State for DNA Bending by IHF

27 pM for the wt IHF–H′ complex (Table 1). Thus, two of the mutants (αK5A and βK84A) exhibited significant loss of stabilizing interactions with bound H′ substrate, while no loss of stability was detected for the αR21C–H′ complex. Equilibrium FRET efficiency values were determined as a function of temperature for αK5A–H′ and βK84A–H′ complexes at two different IHF–DNA concentrations of 1 μM:1 μM and 20 μM:20 μM. For both complexes, the decrease in FRET efficiency with increasing temperature was independent of concentration, indicating that this decrease was primarily from unbending of DNA within the complex and not from any significant bimolecular disruption (Fig. 8b). For αK5A–H′, the FRET value at 20 °C is ∼ 0.25, in comparison with ∼ 0.55 for the wt IHF–H′ complex, indicating that a mutation at this site has a significant effect on the ability of the protein to keep the DNA in a fully bent conformation. For βK84A–H′, the FRET value at 20 °C is ∼ 0.45. Kinetics measurements on all three mutants yielded less than 1.5-fold change in the DNA bending rates in comparison with the wt-IHF (Fig. 8c and Table 1), despite the N 500-fold decrease in binding affinity of two of the mutants for H′. Thus, a change in the free energy of the complex as a result of the destabilizing mutations is not reflected in the free energy of the transition state. We conclude that the transition-state ensemble lacks the stabilizing interactions between the partially bent DNA and the charged residues at the locations αK5 and βK84.

Discussion Dynamics of DNA-bending reveal the underlying energy landscape of binding site recognition by IHF The kinetics of DNA bending reported here reveal the energetics of the IHF–DNA complex by examining how these kinetics are influenced by modifications that render the DNA more flexible at key sites (e.g., NickC and TT8AT) or perturb specific protein–DNA interactions (e.g., H′44A). We also examined how these kinetics are influenced by mutations in IHF that modify the positively charged wrapping surface of the protein. Our results reveal the nature of the transition-state ensemble separating the nonspecific from the specific complex. A nick or mismatches in the DNA at the sites of the kinks relieve bending strain in the transition state for complex formation Crystallographic studies of the IHF–NickC complex illustrated that a nick in the sugar-phosphate backbone directly at the site of one of the kinks allows relief of strain in the bent DNA in a manner

309 very similar to how single-T insertions at the kink sites do so in an HU–DNA complex. 61 If all of the 4fold increase in the binding affinity for NickC in comparison with H′ is attributed to this relief of the bending strain, then we estimate that the nick relieves ∼ 0.8 kcal/mol of strain. 61 Our observation that the bending rate measured for NickC increases by nearly the same factor as its binding affinity suggests that the nicked DNA is already in its relaxed conformation in the transition state, thus lowering the free-energy barrier for DNA bending by the same amount as the release in strain energy. It is important to note that the effect of nicks on the binding affinity and the bending rates is modest and that the apparent activation enthalpy for the bending of the NickC substrate is very similar to that for the intact H′ DNA (Fig. 6c). These results are consistent with previous studies that showed essentially straight conformation for nicked DNA in solution and underscore the importance of stacking in maintaining the helical conformation. 66–69 For the IHF–TT8AT complex, we observe the same trend as for the IHF–NickC complex: an increase in binding affinity is mirrored in accelerated DNA bending rates (Fig. 7b). This acceleration is observed whether we introduce mismatches at or near both kink sites or just one or the other (Fig. 7c), indicating that both arms of the DNA are bent and that the DNA adopts at least a partially “U-bent” conformation in the transition-state ensemble. The effects of modifying protein–DNA contacts distal to the kink sites Kinetics measurements on the IHF–H′44A complex provide a useful control for our picture of the transition-state ensemble. The modification introduced in the H′ sequence in the TTR consensus region alters the “twistability” of DNA whereby disrupting stabilizing ionic interactions between IHF and DNA and illustrates another indirect readout feature that IHF exploits to recognize a part of its binding site. 57,58 This modification is separated from the location of the nearest kink by 6 bp (Fig. 2) and hence perturbs the IHF–DNA interactions without changing the bendability of DNA at the site of the kinks. Our results on the IHF–H′44A complex, which show no change in the DNA bending rates in comparison with the IHF–H′ complex (Fig. 7b and Table 1), demonstrate that the free energy of the transition state remains unchanged. We conclude that the stabilizing interactions between the TTR region and the IHF protein are not made in the transition-state ensemble. In the case of IHF mutants in which positively charged residues in the DNA-wrapping surface are mutated to neutral residues (Fig. 8a), no change in the DNA bending rates is observed despite a N 500fold decrease in binding affinity (Fig. 8c and Table 1).

310

Fig. 9. Schematic representation of the free-energy profile for the IHF–DNA complex. The picture illustrates how the changes in the free energy of the final complex, as a result of modifications in the H′ or IHF sequence, are reflected in the changes in the free energy of the transition state. Nicks (NickC) or mismatches (TT8AT) at the site of the kinks stabilize the complex and the transition state by approximately the same amount, while modifications in the H′ sequence away from the kinks (H′44A) or in the protein (IHF mutants αK5A or βK84A) that destabilize the complex do not perturb the transition state. Whether the β-arms of the protein are wrapped around the DNA with the prolines intercalated at the kinks, in the transition-state ensemble, is not known.

Previously, we demonstrated that the DNA bending step in the IHF–H′ complex is independent of the ionic strength in the solution, which suggested that no ions are released in this rate-limiting step. 50 The results presented here on the IHF mutants corroborate our earlier conclusion and indicate that electrostatic interactions between the wrapping surface of the protein and DNA do not contribute to the rate at which DNA bends in the complex, again pointing to spontaneous thermal fluctuations in DNA as the dominant bottleneck in the transition pathway to form the specific complex. Indirect readout mechanism for binding site recognition Our measurements on the conformational dynamics of the IHF–H′ complex and its variants provide direct evidence for the scenario we proposed in our earlier studies, 49,50 that thermal fluctuations in DNA to adopt “prebent” conformations present the primary bottleneck in binding site recognition by IHF (Fig. 9), with the bent conformation subsequently captured by the protein by a myriad of stabilizing protein–DNA interactions. Theoretical extensions of the continuum, elastic worm-like chain model to describe the mechanical properties of DNA include localized bending/kinking of DNA

Transition State for DNA Bending by IHF

when constrained to form tight circles. 70–72 Such kinked conformations are observed in molecular dynamics simulations of small circular DNA segments. 73 The theoretical and computational studies were motivated by measurements that reported enhanced probabilities for b105-bp-long DNA fragments to form small circles. 74–76 Our results suggest that localized kinking from thermal fluctuations may be an underlying mechanism for binding site recognition even in the absence of such severe distortions. Based on these results, we propose the following mechanism for binding site recognition by DNAbending proteins and, in particular, the minor groove binding proteins that rely primarily on indirect readout: random conformational fluctuations in DNA, which are sequence dependent, allow the DNA to sample conformational space; when these random conformations adopted by the DNA at least partially match the binding interface of the protein, the nonspecifically bound protein recognizes and captures the bent or kinked DNA to form the tightly bound complex. Are prebent DNA conformations thermally accessible in the absence of bound IHF? The analysis of the relaxation kinetics presented here is within the framework of a minimal kinetic scheme of Fig. 3 that assumes a nonspecific complex in which the DNA is straight and a specific complex in which the DNA is bent. However, our observation that the unimolecular kinetics deviate from single-exponential decays indicates that this scheme is at best oversimplified and that additional states will be necessary for a more complete description. In particular, our simple scheme does not include the possibility that prebent DNA conformations for specific sequences are thermally accessible in the absence of bound protein, with IHF binding preferentially to partially bent DNA. A more elaborate scheme that includes this and other possible pathways, including the possibility of the protein fluctuating between two alternate conformations, is illustrated in Fig. 10. The data at the present time are not sufficient to rule in or eliminate any of the states and pathways shown in the scheme of Fig. 10 for binding site recognition by IHF. There is indirect evidence from studies on other DNA-bending proteins that suggests that prebent DNA conformations may be present in rapid pre-equilibrium with straight BDNA conformations for specific sequences that are bent in complex with the protein. One such example is the catabolite-activator protein (CAP) bound to its cognate DNA substrate. Molecular dynamics simulations on the CAP–DNA system, together with simulations of the free protein and DNA, indicated partially prebent DNA conformations in the

311

Transition State for DNA Bending by IHF

out for the CAP–DNA system, together with measurements of distributions of bent conformations in unbound DNA, will be necessary to further shed light on whether prebent DNA conformations in the absence of bound protein are important for binding site recognition by IHF. Binding site recognition versus “sliding” of protein on DNA Fig. 10. A proposed mechanism for the IHF–DNA complex formation. Plausible alternative pathways for the formation of the specific complex, starting from free protein and DNA, are shown for binding site recognition by IHF. The pathways include sequence-dependent, partially prebent DNA conformations in the absence of bound IHF (bottom left) or with IHF bound but prior to conformational changes in IHF necessary for the induced fit (bottom center). A complex in which the DNA is bent in a nonspecific (sequence independent) manner, illustrated by a smooth bend (top right), but with IHF conformation altered, may exist along the transition pathway.

ensemble of conformational states accessible to the unbound DNA. 77 Another piece of evidence comes from measurements of the bimolecular association rates of about 10 6 M − 1 s − 1 for the binding of the TATA-binding protein and a DNA repair protein (MutS) to their target DNA sites, 34,38,78 which are 100- to 1000-fold smaller than the estimated rates of 10 8 M − 1 s − 1 to 10 9 M − 1 s − 1 for diffusion-controlled bimolecular association between protein and a short DNA oligomer. 79,80 Plausible mechanisms that have been proposed to account for this reduction in bimolecular association rates include the possibility that the protein binds only to the fraction of DNA that is partially prebent 35 or, alternatively, that the protein fluctuates between distinct conformations, with only a subset that is able to bind DNA. 81 For the IHF–H′ complex, the measured bimolecular association rate is ∼ 5 × 10 8 M − 1 s − 1, 48 indicating that thermal fluctuations of the cognate DNA site to adopt prebent conformations in the absence of bound IHF may not be a significant factor for the overall association. However, this conclusion presumes that estimates of diffusion-controlled rates have accurately taken into account orientation and electrostatic factors that may affect bimolecular association rates. 82 As an example, removal of the positively charged C-terminal subdomains of a restriction endonuclease (EcoRV) reduces the bimolecular association rate from 1.1 × 10 8 M − 1 s − 1 to 7.5 × 10 4 M − 1 s − 1, 83 indicating a significant contribution from electrostatic complementarity between protein and DNA to the measured association rates. Thus, accurate estimates of true diffusion limited association rates for the IHF–DNA complex and molecular dynamics simulations of the kind carried

We note that IHF–H′ is the only protein–DNA complex for which the unimolecular bending step has been resolved and which has been observed even on the millisecond timescales of stopped-flow measurements. It is possible that the rather severe bend induced in DNA by IHF, together with the fact that the DNA must kink at two sites, makes for the relatively slow recognition of its binding site. It remains to be determined how rapidly the DNA bends in complex with other DNA-bending proteins. An outstanding question remains: how fast must the recognition step be in comparison with the time that a nonspecifically bound protein spends in the vicinity of a potential binding site? Direct visualization of protein sliding on DNA has yielded one-dimensional diffusion constants in the range of ∼ 2 × 10 3 bp 2/s to 7 × 10 6 bp 2/s (reviewed by Barsky et al. 84), which indicate stepping times per base pair of ∼ 140 ns to ∼ 500 μs. Sequence-specific DNA binding proteins need to recognize their target sites faster than the time it takes for them to diffuse away. Thus, for most proteins, this initial recognition step must happen on submillisecond timescales. The fast time resolution of laser T-jump techniques provides an exciting opportunity to unveil this step for other site-specific proteins.

Methods Materials All DNA oligonucleotides were synthesized and PAGE purified by the W. M. Keck facility at Yale University. The oligonucleotides were labeled with fluorescein (F) and TAMRA (T) by incorporating F-dT and R-dT (Glen Research, Sterling, VA) at the 5′ ends of the top and bottom strands, respectively, as indicated in Fig. 2. The concentrations of each DNA strand were determined by measuring the UV absorbance at 260 nm using the extinction coefficients 4.00 × 10 5 M − 1 cm − 1 for the fluorescein-labeled top strand and 4.15 × 10 5 M − 1 cm − 1 for the TAMRA-labeled bottom strand. 60 For determination of the amount of labeled DNA in solution, fluorescein and TAMRA concentrations were also determined in the labeled samples by measuring the absorbance of F-labeled strands at 494 nm and R-labeled strands at 555 nm and compared with the concentrations of the oligomers obtained from measurements at 260 nm. For the dyes, the molar extinction coefficients were 75,000 M − 1cm − 1 for

312 F-dT at 494 nm and 89,000 M − 1cm − 1 for R-dT at 556 nm (Molecular Probes§). The percentage of unlabeled DNA in solution was estimated to be ∼ 5–10%. Both IHF subunits were cloned as a single operon in a pET21a vector, and mutations were introduced using the QuikChange kit (Strategene). The proteins were purified essentially as described previously, 58 using (NH4)2SO4 cuts followed by heparin and mono S chromatography. All buffers used with αR21C included 2 mM DTT. Mutant proteins were expressed in derivatives of Escherichia coli strain BL21(DE3) lacking functional genes for the mutated subunit (strains K1299 and JG1244 were gifts from Jeffrey Gardner, University of Illinois at Urbana-Champaign). Proteins were checked for nuclease activity by incubating with supercoiled plasmid and 5 mM MgCl2 and rechromatographed with slower gradients, if necessary. IHF proteins were stored at 454 μM in a storage buffer of 25 mM Hepes (pH 7.5), 200 mM NaCl, 20% glycerol, and 1 mM ethylenediaminetetraacetic acid (EDTA). Droplets of proteins were first flash-frozen in liquid nitrogen prior to storage in cryogenic tubes at −80 °C. Individual frozen droplets were diluted into binding buffer [20 mM Tris– HCl, 1 mM EDTA (pH 8), plus 2 mM DTT in αR21C samples, and KCl concentrations from 100 mM to 500 mM]. IHF concentrations were measured by absorbance at 276 nm, with an extinction coefficient of 5800 M − 1 cm − 1. 58 Equilibrium measurements The steady-state fluorescence emission spectra were measured on a FluoroMax2 spectrofluorimeter (Jobin Yvon, Inc., NJ). The FRET efficiency and acceptor ratio values were obtained from the measured spectra under each condition, as described in Supplementary Text. The errors are about ±0.05 in the FRET efficiency measurements and about ± 0.01 in the acceptor ratio measurements. The dissociation constants were obtained from salt titration experiments in which the acceptor ratio for each complex was measured for varying [KCl] concentrations, as described in Supplementary Text. The uncertainties in the Kd and SKd values reported in Table 1 are from statistical variations in the fitting parameters, from at least two sets of independent measurements. Laser T-jump spectrometer Rapid T-jump was achieved in sample cuvettes of path length 0.5 mm, as described previously. 49,50 For measurements on the IHF–NickC complex and for some of the earlier measurements on the IHF–H′ complex, the donor fluorescence was excited by a 200-W Hg-Xe lamp, with the excitation wavelengths selected by a broadband filter with transmission in the range of 440–490 nm. For measurements on all other complexes, the donor fluorescence was excited with a 20-mW cw diode laser at 488 nm (Newport PC13589). The fluorescence emission intensity was monitored perpendicular to the excitation direction, with a combination of a long pass filter (N 490 nm) and a short pass filter (b 550 nm), and measured with a

§ www.glenresearch.com/Technical/Extinctions.html

Transition State for DNA Bending by IHF

Hamamatsu R928 photomultiplier tube. The magnitude of the T-jump was determined as described in Vivas et al. 50 The errors in the T-jump estimates are about 10–20%.

Acknowledgements We dedicate this article to Jonathan Widom, who was and remains a source of inspiration and whose profound insight, thoughtful comments, and encouragement, over the years, have been invaluable. We thank Sawako Sugimura and Donald Crothers (Yale University) for sharing the results of their stopped-flow measurements on the IHF–NickC complex, Ying Z. Pigli (University of Chicago) for purification of the mutant IHF proteins, and Cierra Hall for help with the analysis of equilibrium titration measurements. A.A. acknowledges support from the National Science Foundation (MCB0721937). S.V.K. acknowledges support from the American Heart Association (AHA 0730254N).

Supplementary Data Supplementary data to this article can be found online at doi:10.1016/j.jmb.2012.02.028

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