Serguei V. Kuznetsov

Configurational diffusion down a folding funnel describes the dynamics of DNA hairpins

Elucidating the mechanism of folding of polynucleotides depends on accurate estimates of free energy surfaces and a quantitative description of the kinetics of structure formation. Here, the kinetics of hairpin formation in single-stranded DNA are measured after a laser temperature jump. The kinetics are modeled as configurational diffusion on a free energy surface obtained from a statistical mechanical description of equilibrium melting profiles. The effective diffusion coefficient is found to be strongly temperature-dependent in the nucleation step as a result of formation of misfolded loops that do not lead to subsequent zipping. This simple system exhibits many of the features predicted from theoretical studies of protein folding, including a funnel-like energy surface with many folding pathways, trapping in misfolded conformations, and non-Arrhenius folding rates.

Loop dependence of the stability and dynamics of nucleic acid hairpins

Hairpin loops are critical to the formation of nucleic acid secondary structure, and to their function. Previous studies revealed a steep dependence of single-stranded DNA (ssDNA) hairpin stability with length of the loop (L) as ∼L8.5 ± 0.5, in 100 mM NaCl, which was attributed to intraloop stacking interactions. In this article, the loop-size dependence of RNA hairpin stabilities and their folding/unfolding kinetics were monitored with laser temperature-jump spectroscopy. Our results suggest that similar mechanisms stabilize small ssDNA and RNA loops, and show that salt contributes significantly to the dependence of hairpin stability on loop size. In 2.5 mM MgCl2, the stabilities of both ssDNA and RNA hairpins scale as ∼L4 ± 0.5, indicating that the intraloop interactions are weaker in the presence of Mg2+. Interestingly, the folding times for ssDNA hairpins (in 100 mM NaCl) and RNA hairpins (in 2.5 mM MgCl2) are similar despite differences in the salt conditions and the stem sequence, and increase similarly with loop size, ∼L2.2 ± 0.5 and ∼L2.6 ± 0.5, respectively. These results suggest that hairpins with small loops may be specifically stabilized by interactions of the Na+ ions with the loops. The results also reinforce the idea that folding times are dominated by an entropic search for the correct nucleating conformation.

Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

Regulation of gene expression involves formation of specific protein–DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNA-binding sites. Previous kinetics measurements on several DNA-bending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending/unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from ≈2 ms−1 at ≈37°C to ≈40 ms−1 at ≈10°C and correspond to an activation energy of ≈14 ± 3 kcal/mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend/kink DNA before forming a tight complex with IHF.

New Insights into the Transition Pathway from Nonspecific to Specific Complex of DNA with Escherichia Coli Integration Host Factor

To elucidate the nature of the transition-state ensemble along the reaction pathway from a nonspecific protein-DNA complex to the specific complex, we have carried out measurements of DNA bending/unbending dynamics on a cognate DNA substrate in complex with integration host factor (IHF), an architectural protein from E. coli that bends its cognate site by approximately 180 degrees . We use a laser temperature jump to perturb the IHF-DNA complex and monitor the relaxation kinetics with time-resolved FRET measurements on DNA substrates end-labeled with a FRET pair. Previously, we showed that spontaneous bending/kinking of DNA, from thermal disruption of base-pairing/-stacking interactions, may be the rate-limiting step in the formation of the specific complex (Kuznetsov, S. V.; Sugimura, S.; Vivas, P.; Crothers, D. M.; Ansari, A. Proc. Natl. Acad. Sci. USA 2006, 103, 18515). Here, we probe the effect of varying [KCl], which affects the stability of the complex, on this rate-limiting step. We find that below approximately 250 mM KCl, the observed relaxation kinetics are from the unimolecular bending/unbending of DNA, and the relaxation rate kr is independent of [KCl]. Above approximately 300 mM KCl, dissociation of the IHF-DNA complex becomes significant, and the observed relaxation process includes contributions from the association/dissociation step, with kr decreasing with increasing [KCl]. The DNA bending step occurs with a positive activation enthalpy, despite the large negative enthalpy change reported for the specific IHF-DNA complex (Holbrook, J. A.; Tsodikov, O. V.; Saecker, R. M.; Record, M. T., Jr. J. Mol. Biol. 2001, 310, 379). Our conclusion from these studies is that in the uphill climb to the transition state, the DNA is kinked, but with no release of ions, as indicated by the salt-independent behavior of k(r) at low [KCl]. Any release of ions in the unimolecular process, together with conformational changes in the protein-DNA complex that facilitate favorable interactions and that contribute to the negative enthalpy change, must occur as the system leaves the transition state, downhill to the final complex.

Fast folding of RNA pseudoknots initiated by laser temperature-jump.

RNA pseudoknots are examples of minimal structural motifs in RNA with tertiary interactions that stabilize the structures of many ribozymes. They also play an essential role in a variety of biological functions that are modulated by their structure, stability, and dynamics. Therefore, understanding the global principles that determine the thermodynamics and folding pathways of RNA pseudoknots is an important problem in biology, both for elucidating the folding mechanisms of larger ribozymes as well as addressing issues of possible kinetic control of the biological functions of pseudoknots. We report on the folding/unfolding kinetics of a hairpin-type pseudoknot obtained with microsecond time-resolution in response to a laser temperature-jump perturbation. The kinetics are monitored using UV absorbance as well as fluorescence of extrinsically attached labels as spectroscopic probes of the transiently populated RNA conformations. We measure folding times of 1-6 ms at 37 °C, which are at least 100-fold faster than previous observations of very slow folding pseudoknots that were trapped in misfolded conformations. The measured relaxation times are remarkably similar to predictions of a computational study by Thirumalai and co-workers (Cho, S. S.; Pincus, D.L.; Thirumalai, D. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 17349-17354). Thus, these studies provide the first observation of a fast-folding pseudoknot and present a benchmark against which computational models can be refined.

Exploring the energy landscape of nucleic acid hairpins using laser temperature-jump and microfluidic mixing.

We have investigated the multidimensionality of the free energy landscape accessible to a nucleic acid hairpin by measuring the relaxation kinetics in response to two very different perturbations of the folding/unfolding equilibrium, either a laser temperature-jump or ion-jump (from rapid mixing with counterions). The two sets of measurements carried out on DNA hairpins (4 or 5 base pairs in the stem and 21-nucleotide polythymine loop), using FRET between end labels or fluorescence of 2-aminopurine in the stem as conformational probes, yield distinctly different relaxation kinetics in the temperature range 10-30 °C and salt range 100-500 mM NaCl, with rapid mixing exhibiting slower relaxation kinetics after an initial collapse of the chain within 8 μs of the counterion mixing time. The discrepancy in the relaxation times increases with increasing temperatures, with rapid mixing times nearly 10-fold slower than T-jump times at 30 °C. These results rule out a simple two-state scenario with the folded and unfolded ensemble separated by a significant free energy barrier, even at temperatures close to the thermal melting temperature T(m). Instead, our results point to the scenario in which the conformational ensemble accessed after counterion condensation and collapse of the chain is distinctly different from the unfolded ensemble accessed with T-jump perturbation. Our data suggest that, even at temperatures in the vicinity of T(m) or higher, the relaxation kinetics obtained from the ion-jump measurements are dominated by the escape from the collapsed state accessed after counterion condensation.

A kinetic zipper model with intrachain interactions applied to nucleic acid hairpin folding kinetics.

Single-stranded DNA and RNA hairpin structures with 4-10 nucleotides (nt) in the loop and 5-8 basepairs (bp) in the stem fold on 10-100 μs timescale. In contrast, theoretical estimate of first contact time of two ends of an ideal semiflexible polymer of similar lengths (with persistence length ~2-nt) is 10-100 ns. We propose that this three-orders-of-magnitude difference between these two timescales is a result of roughness in the folding free energy surface arising from intrachain interactions. We present a statistical mechanical model that explicitly includes all misfolded microstates with nonnative Watson-Crick (WC) and non-WC contacts. Rates of interconversion between different microstates are described in terms of two adjustable parameters: the strength of the non-WC interactions (ΔG(nWC)) and the rate at which a basepair is formed adjacent to an existing basepair (k(bp)(+)). The model accurately reproduces the temperature and loop-length dependence of the measured relaxation rates in temperature-jump studies of a 7-bp stem, single-stranded DNA hairpin with 4-20-nt-long poly(dT) loops, with ΔG(nWC) ≈ -2.4 kcal/mol and k(bp)(+) ≥ (1 ns)(-1), in 100 mM NaCl. Thus, our model provides a microscopic interpretation of the slow hairpin folding times as well as an estimate of the strength of intrachain interactions.

Dynamics and Mechanism of DNA-Bending Proteins in Binding Site Recognition

The three-dimensional shape of biological macromolecules (proteins, DNA, and RNA), is determined by a myriad of “weak” noncovalent interactions (ionic, hydrophobic, van der Waals, and hydrogen bonds), each of which can be disrupted by thermal fluctuations, leading to constantly changing conformations accessible to the macromolecule [1]. These conformational fluctuations are essential to biology and are central to molecular recognition, in which two or more interacting macromolecules rely on complementary shapes and charge distributions to form a multitude of weak intermolecular bonds that lead to higher-order complexes. An overarching goal in molecular biophysics is to elucidate the underlying energetics of these interactions, by measuring the dynamics of conformational fluctuations in the macromolecular complexes.

Global analysis of ion dependence unveils hidden steps in DNA binding and bending by integration host factor

Proteins that recognize and bind to specific sites on DNA often distort the DNA at these sites. The rates at which these DNA distortions occur are considered to be important in the ability of these proteins to discriminate between specific and nonspecific sites. These rates have proven difficult to measure for most protein-DNA complexes in part because of the difficulty in separating the kinetics of unimolecular conformational rearrangements (DNA bending and kinking) from the kinetics of bimolecular complex association and dissociation. A notable exception is the Integration Host Factor (IHF), a eubacterial architectural protein involved in chromosomal compaction and DNA recombination, which binds with subnanomolar affinity to specific DNA sites and bends them into sharp U-turns. The unimolecular DNA bending kinetics has been resolved using both stopped-flow and laser temperature-jump perturbation. Here we expand our investigation by presenting a global analysis of the ionic strength dependence of specific binding affinity and relaxation kinetics of an IHF-DNA complex. This analysis enables us to obtain each of the underlying elementary rates (DNA bending/unbending and protein-DNA association/dissociation), and their ionic strength dependence, even under conditions where the two processes are coupled. Our analysis indicates interesting differences in the ionic strength dependence of the bi- versus unimolecular steps. At moderate [KCl] (100-500 mM), nearly all the ionic strength dependence to the overall equilibrium binding affinity appears in the bimolecular association/dissociation of an initial, presumably weakly bent, encounter complex, with a slope SK(bi) ≈ 8 describing the loglog-dependence of the equilibrium constant to form this complex on [KCl]. In contrast, the unimolecular equilibrium constant to form the fully wrapped specific complex from the initial complex is nearly independent of [KCl], with SK(uni) < 0.5. This result is counterintuitive because there are at least twice as many ionic protein-DNA contacts in the fully wrapped complex than in the weakly bent intermediate. The following picture emerges from this analysis: in the bimolecular step, the observed [KCl]-dependence is consistent with the number of DNA counterions expected to be released when IHF binds nonspecifically to DNA whereas in the unimolecular reorganization step, the weak [KCl]-dependence suggests that two effects cancel one another. On one hand, formation of additional protein-DNA contacts in the fully wrapped complex releases bound counterions into bulk solution, which is entropically favored by decreasing [salt]. On the other hand, formation of the fully wrapped complex also releases tightly bound water molecules, which is osmotically favored by increasing [salt]. More generally, our global analysis strategy is applicable to other protein-DNA complexes, and opens up the possibility of measuring DNA bending rates in complexes where the unimolecular and bimolecular steps are not easily separable.

Hairpin Formation in Polynucleotides: A Simple Folding Problem?

The biological processes in cell involving DNA, e.g. replication, recombination, DNA repair and transcription, are accompanied by localized unwinding of the DNA molecule. This unwinding may occur as a result of fluctuational opening of individual base-pairs (Wartell and Benight, 1985), or can be driven by DNA binding proteins, leading to the formation of single-stranded (ss) region(s) in DNA (Lohman and Bjornson, 1996; Bianco et al., 2001). Palindromic regions of unwinded DNA can form irregular structures by base-pairing between neighboring self-complementary sequences. These structures are known as cruciforms and hairpins. The formation of hairpins and their involvement in biological processes such as replication and transcription is now well documented in both prokaryotic and eukaryotic systems (Crews et al., 1979; Wilson and von Hippel, 1995; Glucksmann-Kuis et al., 1996; Dai et al., 1997). It has been shown that DNA hairpins can serve as intermediates in genetic recombination (Lilley, 1981; Romer et al., 1984; Roth et al., 1992), and as protein recognition sites, and can regulate transcription in vivo (Dai et al., 1997; Dai et al., 1998). The biological and biomedical significance of hairpin structures is also associated with the fact that the formation of DNA hairpins by GC-rich triplet repeat, and inordinate expansion of these triplets during DNA replication, leads to several genetic diseases including a progressive neuromuscular disorder (Caskey et al., 1992; Gacy et al., 1995). Short ssDNA and RNA hairpin structures are also useful drug targets because their overall shape and geometry differ significantly from regular double-stranded DNA, and