Time-resolved infrared spectroscopy of RNA folding

We introduce time-resolved infrared spectroscopy as a powerful method to study the kinetics of RNA folding and unfolding transitions. A laser-induced temperature jump is used to initiate a perturbation in the RNA structure. A probe laser, tuned to a specific infrared absorption of the RNA, is then used to monitor the subsequent relaxation kinetics. A 10-ns pump pulse permits the investigation of fast, nanosecond events. In this work we probe two vibrational transitions, one at 1620 cm(-1) and one at 1661 cm(-1). The former transition reports mainly on the dynamics of A and U interactions, the latter is attributed to mainly G and C interactions. Our results reveal three distinct kinetic phases for each vibrational transition probed. We propose two models to describe the data. In one mechanism, the unfolded state partitions into two separate populations; each is conformationally biased to proceed via one of two distinct pathways. In an alternative model, folding proceeds through a series of sequentially populated intermediates. In both cases, the first step in the proposed folding mechanism is rate limiting (hundreds of microseconds) and involves a collapse into incorrectly folded intermediate populations. Two faster kinetic phases (tens of microseconds and hundreds of nanoseconds) follow in which the intermediate populations undergo localized reorganizational motions in the search for native contacts.     

Probing the folding and unfolding dynamics of secondary and tertiary structures in a three-helix bundle protein

Fast relaxation kinetics studies of the B-domain of staphylococcal protein A were performed to characterize the folding and unfolding of this small three-helix bundle protein. The relaxation kinetics were initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The kinetics monitored within the amide I' absorbance of the polypeptide backbone exhibit two distinct kinetics phases with nanosecond and microsecond relaxation times. The fast kinetics relaxation time is close to the diffusion limits placed on protein folding reactions. The fast kinetics phase is dominated by the relaxation of the solvated helix (nu = 1632 cm(-1)), which reports on the fast relaxation of the individual helices. The slow kinetics phase follows the cooperative relaxation of the native helical bundle core that is monitored by both solvated (nu = 1632 cm(-1)) and buried helical IR bands (nu = 1652 cm(-1)). The folding rates of the slow kinetics phase calculated over an extended temperature range indicate that the core formation of this protein follows a pathway that is energetically downhill. The unfolding rates are much more strongly temperature-dependent indicating an activated process with a large energy barrier. These results provide significant insight into the primary process of protein folding and suggest that fast formation of helices can drive the folding of helical proteins.     

The dynamic structural basis of differential enhancement of conformational stability by 5'- and 3'-dangling ends in RNA

Unpaired bases at the end of an RNA duplex (dangling ends) can stabilize the core duplex in a sequence-dependent manner and are important determinants of RNA folding, recognition, and functions. Using 2-aminopurine as a dangling end purine base, we have employed femtosecond time-resolved fluorescence spectroscopy, combined with UV optical melting, to quantitatively investigate the physical and structural nature of the stacking interactions between the dangling end bases and the terminal base pairs. A 3'-dangling purine base has a large subpopulation that stacks on the guanine base of the terminal GC or UG pair, either intrastrand or cross-strand depending on the orientation of the pair, thus providing stabilization of different magnitudes. On the contrary, a 5'-dangling purine base only has a marginal subpopulation that stacks on the purine of the same strand (intrastrand) but has little cross-strand stacking. Thus a 5'-dangling purine does not provide significant stabilization. These stacking structures are not static, and a dangling end base samples a range of stacked and unstacked conformations with respect to the terminal base pair. Femtosecond time-resolved anisotropy decay reveals certain hindered base conformational dynamics that occur on the picosecond to nanosecond time scales, which allow the dangling base to sample these substates. When the dangling purine is opposite to a U and is able to form a potential base pair at the end of the duplex, there is an interplay of base stacking and hydrogen-bonding interactions that depends on the orientation of the base pair relative to the adjacent GC pair. By resolving these populations that are dynamically exchanging on fast time scales, we elucidated the correlation between dynamic conformational distributions and thermodynamic stability.     

Some hydrodynamic and optical properties of polyribonucleotides

The size and shape of four polyribonucleotides were studied by sedimentation and viscosity measurements. The results were correlated with their conformations based on optical activity studies. Polyriboadenylic acid in acidic solution dimerizes into two double-stranded forms, one with half-protonated bases and the other fully protonated. The fully protonated form is somewhat less asymmetrical than the half-protonated form. Polyriboguanylic acid in alkaline solution (near the second pK_a of guanine) undergoes a time-dependent disaggregation and the final form shows little base stacking. In acidic solution, it demonstrates a reversible transition near the first pK_a of guanine but without evidence of disaggregation. Polyribocytidylic acid undergoes a transition upon half protonation of the bases, but its molecular weight remains unchanged with pH. The results suggest that this polymer assumes a hairpin structure in acidic solution. Polyribouridylic acid has some degree of base stacking: at room temperature. A transition to a hairpin structure occurs at low temperature.     

On the stability of nucleic acid structures in solution: enthalpy-entropy compensations, internal rotations and reversibility.

The thermodynamics of self-association (stacking) of free bases and nucleotides, intramolecular stacking in dinucleotides, nearest-neighbour base pair stacking interactions in duplex DNA and RNA, and the formation of hairpin loops illustrate enthalpy/entropy compensations. Large stacking exothermicities are associated with large negative entropy changes that ensure that delta G is small, permitting readily reversible associations in solution. We rationalise enthalpy/entropy compensations with reference to residual motions and torsional vibrations which make a larger entropic contribution to binding when - delta H approximately kT (thermal energy at room temperature), than when - delta H >> kT. We present a factorisation of experimental free energies for helix formation in terms of approximate contributions from the restriction of rotations, hydrophobic interactions, electrostatic interactions due to base stacking, and contributions from hydrogen bonding, and estimate the adverse free energy cost per rotor (mainly entropy) of ordering the phosphate backbone as between 1.9 and 5.4 kJ mol-1 [averaged over 12 rotors per base pair for A-U on A-U stacking (lower limit), and G-C on C-G stacking (upper limit)]. The largest cost is associated with the most exothermic stacking interactions, while the range of values is consistent with earlier conclusions from data on the fusion of hydrocarbon chains (lower value), and with entropy changes in covalent isomerisations of small molecules involving severe restrictions (upper value).     

Binding effects of Mn2+ and Zn2+ ions on the vibrational properties of guanine-cytosine base pairs in the Watson-Crick and Hoogsteen configurations

The binding effects of Mn(2+) and Zn(2+) ions on the vibrational properties of guanine-cytosine base pairs have been performed using density functional theory investigations. The calculations were carried out on Watson-Crick and Hoogsteen configurations of the base pairs. We have found, that in Watson-Crick configuration, the metal is coordinated to N7 atom of guanine while, in the case of Hoogsteen configuration, the coordination is at N3 atom of guanine. We have pointed out the vibrational bands that can be used to detect the presence of metallic ions in the Watson-Crick and Hoogsteen structures. Our results show that the vibrational amplitudes of metallic atoms are strong for wavenumbers lower than 600?cm(-1). Also, we predict that the distinction between Watson-Crick and Hoogsteen configurations can be seen around 85, 170 and 310?cm(-1).     

Loop and stem dynamics during RNA hairpin folding and unfolding

2-Aminopurine (2AP) is a fluorescent adenine analog that probes mainly base stacking in nucleic acids. We labeled the loop or the stem of the RNA hairpin gacUACGguc with 2AP to study folding thermodynamics and kinetics at both loci. Thermal melts and fast laser temperature jumps detected by 2AP fluorescence monitored the stability and folding/unfolding kinetics. The observed thermodynamic and kinetic traces of the stem and loop mutants, though strikingly different at a first glance, can be fitted to the same free-energy landscape. The differences between the two probe locations arise because base stacking decreases upon unfolding in the stem, whereas it increases in the loop. We conclude that 2AP is a conservative adenine substitution for mapping out the contributions of different RNA structural elements to the overall folding process. Molecular dynamics (MD) totaling 0.6 μsec were performed to look at the conformations populated by the RNA at different temperatures. The combined experimental data, and MD simulations lead us to propose a minimal four-state free-energy landscape for the RNA hairpin. Analysis of this landscape shows that a sequential folding model is a good approximation for the full folding dynamics. The frayed state formed initially from the native state is a heterogeneous ensemble of structures whose stem is frayed either from the end or from the loop.     

Multiple geminate ligand recombinations in human hemoglobin

The geminate ligand recombination reactions of photolyzed carbonmonoxyhemoglobin were studied in a nanosecond double-excitation-pulse time-resolved absorption experiment. The second laser pulse, delayed by intervals as long as 400 ns after the first, provided a measure of the geminate kinetics by rephotolyzing ligands that have recombined during the delay time. The peak-to-trough magnitude of the Soret band photolysis difference spectrum measured as a function of the delay between excitation pulses showed that the room temperature kinetics of geminate recombination in adult human hemoglobin are best described by two exponential processes, with lifetimes of 36 and 162 ns. The relative amounts of bimolecular recombination to T- and R-state hemoglobins and the temperature dependence of the submicrosecond kinetics between 283 and 323 K are also consistent with biexponential kinetics for geminate recombination. These results are discussed in terms of two models: geminate recombination kinetics modulated by concurrent protein relaxation and heterogeneous kinetics arising from alpha and beta chain differences.     

Base dynamics in a UUCG tetraloop RNA hairpin characterized by 15N spin relaxation: correlations with structure and stability.

Intramolecular dynamics of guanine and uracil bases in a 14-nt RNA hairpin including the extraordinarily stable UUCG tetraloop were studied by 15N spin relaxation experiments that are sensitive to structural fluctuations occurring on a time scale of picoseconds to nanoseconds. The relaxation data were interpreted in the framework of the anisotropic model-free formalism, using assumed values for the chemical shift anisotropies of the 15N spins. The rotational diffusion tensor was determined to be symmetric with an axial ratio of 1.34 +/- 0.12, in agreement with estimates based on the ratio of the principal moments of the inertia tensor. The model-free results indicate that the bases of the G x U pair in the tetraloop are at least as rigid as the interior base pairs in the stem, whereas the 5'-terminal guanine is more flexible. The observed range of order parameters corresponds to base fluctuations of 19-22 degrees about the chi torsion angle. The results reveal dynamical consequences of the unusual structural features in the UUCG tetraloop and offer insights into the configurational entropy of hairpin formation.     

A transition from a twice-folded structure to a hairpin in oligo(dG) in the presence of magnesium ions

A long (dG)n stretch can fold twice forming an intramolecular tetrahelix stabilized by guanine tetrads and stacking interactions between them (G-structure). In this paper, we show that magnesium ions induce a transition of the (dG)n stretch from the G-structure to the hairpin stabilized by Hoogsteen-like G.G base pairs (G-hairpin). This transition between the G-structure and the G-hairpin is detected by chemical probing. The characteristic time of the transition for a (dG)28 at 4 degrees C exceeds five hours, which allowed us to separate these two forms by electrophoresis at low temperature. We believe that the slowness of the transition is due to the fact that half of the deoxyguanosines in the insert must change their conformation from syn to anti or vice versa.     

Probing the structure and dynamics of a DNA hairpin by ultrafast quenching and fluorescence depolarization.

DNA hairpins have been investigated in which individual adenines were replaced by their fluorescent analog 2-aminopurine (2AP). The temperature dependence of the time evolution of polarized emission spectra was monitored with picosecond time resolution. Four isotropic decay components for each oligonucleotide indicated the coexistence of at least four conformations. The fluorescence for three of these was significantly quenched, which is explained by hole transfer from 2AP to guanine(s). An approximately 8-ps component is ascribed to direct hole transfer, the approximately 50-ps and approximately 500-ps components are ascribed to structural reorganization, preceding hole transfer. At room temperature, a fraction remains unquenched on a 10-ns timescale, in contrast to higher temperatures, where the flexibility increases. Besides quenching due to base stacking, a second quenching process was needed to describe the data. Evidence for both intrastrand and interstrand hole transfer was found. The extracted probability for stacking between neighboring bases in double-stranded regions was estimated to be approximately 75% at room temperature and approximately 25% at 80 degrees C, demonstrating structural disorder of the DNA. Fluorescence depolarization revealed both local dynamics of the DNA and overall dynamics of the entire oligonucleotide. Upon raising the temperature, the C-N terminus of the hairpin appears to melt first; the rest of the hairpin denatures above the average melting temperature.     

Structural basis of pyrimidine specificity in the MS2 RNA hairpin-coat-protein complex.

We have determined the X-ray structures of six MS2 RNA hairpin-coat-protein complexes having five different substitutions at the hairpin loop base -5. This is a uracil in the wild-type hairpin and contacts the coat protein both by stacking on to a tyrosine side chain and by hydrogen bonding to an asparagine side chain. The RNA consensus sequence derived from coat protein binding studies with natural sequence variants suggested that the -5 base needs to be a pyrimidine for strong binding. The five -5 substituents used in this study were 5-bromouracil, pyrimidin-2-one, 2-thiouracil, adenine, and guanine. The structure of the 5-bromouracil complex was determined to 2.2 A resolution, which is the highest to date for any MS2 RNA-protein complex. All the complexes presented here show very similar conformations, despite variation in affinity in solution. The results suggest that the stacking of the -5 base on to the tyrosine side chain is the most important driving force for complex formation. A number of hydrogen bonds that are present in the wild-type complex are not crucial for binding, as they are missing in one or more of the complexes. The results also reveal the flexibility of this RNA-protein interface, with respect to functional group variation, and may be generally applicable to other RNA-protein complexes.     

Single-strand stacking free energy from DNA beacon kinetics

DNA beacons are short single-stranded chains which can form closed hairpin shapes through complementary base pairing at their ends. Contrary to the common polymer theory assumption that only their loop length matters, experiments show that their closing kinetics depend on the loop composition. We have modeled the closing kinetics and in so doing have obtained stacking enthalpies and entropies for single-stranded nucleic acids. The resulting change of persistence length with temperature effects the dynamics. With a Monte Carlo study, we answer another polymer question of how the closing time scales with chain length, finding tau approximately N(2.44+/-0.02). There is a significant crossover for shorter chains, bringing the effective exponent into good agreement with experiment.     

Recruitment of a foreign quinone into the A1 site of photosystem I. Altered kinetics of electron transfer in phylloquinone biosynthetic pathway mutants studied by time-resolved optical, EPR, and electrometric techniques

Interruption of the menA or menB gene in Synechocystis sp. PCC 6803 results in the incorporation of a foreign quinone, termed Q, into the A(1) site of photosystem I with a number of experimental indicators identifying Q as plastoquinone-9. A global multiexponential analysis of time-resolved optical spectra in the blue region shows the following three kinetic components: 1) a 3-ms lifetime in the absence of methyl viologen that represents charge recombination between P700(+) and an FeS(-) cluster; 2) a 750-microseconds lifetime that represents electron donation from an FeS(-) cluster to methyl viologen; and 3) an approximately 15-microseconds lifetime that represents an electrochromic shift of a carotenoid pigment. Room temperature direct detection transient EPR studies of forward electron transfer show a spectrum of P700(+) Q(-) during the lifetime of the spin polarization and give no evidence of a significant population of P700(+) FeS(-) for t 相似文献   

Gating current kinetics in Myxicola giant axons. Order of the back transition rate constants.

Gating current, Ig, was recorded in Myxicola axons with series resistance compensation and higher time resolution than in previous studies. Ig at ON decays as two exponentials with time constants, tau ON-F and tau ON-S, very similar to squid values. No indication of an additional very fast relaxation was detected, but could be still unresolved. Ig at OFF also displays two exponentials, neither reflecting recovery from charge immobilization. Deactivation of the two I(ON) components may proceed with well-separated exponentials at -100 mV. INa tail currents at OFF also display two exponentials plus a third very slow relaxation of 5-9% of the total tail current. The very slow component is probably deactivation of a very small subpopulation of TTX sensitive channels. A -100 mV, means for INa tail component time constants (four axons) are 76 microseconds (range: 53-89 microseconds) and 344 microseconds (range: 312-387 microseconds), and for IOFF (six axons) 62 microseconds (range: 34-87 microseconds) and 291 microseconds (range: 204-456 microseconds) in reasonable agreement. INa ON activation time constant, tau A, is clearly slower than tau ON-F at all potentials. Except for the interval -30 to -15 mV, tau A is clearly faster than tau ON-S, and has a different dependency on potential. tau ON-S is several fold smaller than tau h. Computations with a closed2----closed1----open activation model indicated Na tail currents are consistent with a closed1----open rate constant greater than the closed2----closed1.     

Core formation in apomyoglobin: probing the upper reaches of the folding energy landscape

An acid-destabilized form of apomyoglobin, the so-called E state, consists of a set of heterogeneous structures that are all characterized by a stable hydrophobic core composed of 30-40 residues at the intersection of the A, G, and H helices of the protein, with little other secondary structure and no other tertiary structure. Relaxation kinetics studies were carried out to characterize the dynamics of core melting and formation in this protein. The unfolding and/or refolding response is induced by a laser-induced temperature jump between the folded and unfolded forms of E, and structural changes are monitored using the infrared amide I' absorbance at 1648-1651 cm(-1) that reports on the formation of solvent-protected, native-like helix in the core and by fluorescence emission changes from apomyoglobin's Trp14, a measure of burial of the indole group of this residue. The fluorescence kinetics data are monoexponential with a relaxation time of 14 micros. However, infrared kinetics data are best fit to a biexponential function with relaxation times of 14 and 59 micros. These relaxation times are very fast, close to the limits placed on folding reactions by diffusion. The 14 micros relaxation time is weakly temperature dependent and thus represents a pathway that is energetically downhill. The appearance of this relaxation time in both the fluorescence and infrared measurements indicates that this folding event proceeds by a concomitant formation of compact secondary and tertiary structures. The 59 micros relaxation time is much more strongly temperature dependent and has no fluorescence counterpart, indicating an activated process with a large energy barrier wherein nonspecific hydrophobic interactions between helix A and the G and H helices cause some helix burial but Trp14 remains solvent exposed. These results are best fit by a multiple-pathway kinetic model when U collapses to form the various folded core structures of E. Thus, the results suggest very robust dynamics for core formation involving multiple folding pathways and provide significant insight into the primary processes of protein folding.     

Fast Folding of an RNA Tetraloop on a Rugged Energy Landscape Detected by a Stacking-Sensitive Probe

We investigate the microsecond-timescale kinetics of the RNA hairpin ga^∗cUUCGguc. The fluorescent nucleotide 2-aminopurine (a^∗) reports mainly on base stacking. Ten kinetic traces and the temperature denaturation curve are globally fitted to four-state models of the free-energy surface. In the best-fitting sequential model, the hairpin unfolds over successively larger barriers in at least three stages: stem fraying and increased base-stacking fluctuations; concerted loss of hydrogen bonding and partial unstacking; and additional unstacking of single strands at the highest temperatures. Parallel and trap models also provide adequate fits: such pathways probably also play a role in the complete free-energy surface of the hairpin. To interpret the model states structurally, 200 ns of molecular dynamics, including six temperature-jump simulations, were run. Although the sampling is by no means comprehensive, five different states were identified using hydrogen bonding and base stacking as reaction coordinates. The four to five states required to explain the experiments or simulations set a lower limit on the complexity of this small RNA hairpin's energy landscape.     

Dynamics of human serum albumin studied by acoustic relaxation spectroscopy

Sonic absorption spectra of solutions of human serum albumin (SA) in water and in aqueous phosphate buffer systems have been measured between 0.2 and 2000 MHz at different temperatures (15-35 degrees C), pH values (1.8-12.3), and protein concentrations (1-40 g/L). Several spectra, indicating relaxation processes in the whole frequency range, have been found. The spectra at neutral pH could be fitted well with an analytical function consisting of the asymptotic high frequency absorption and two relaxation contributions, a Debye-type relaxation term with discrete relaxation time and a term with asymmetric continuous distribution of relaxation times. Both relaxation contributions were observed in water and in buffer solutions and increased with protein concentration. The contribution represented by a Debye-type term is practically independent of temperature and was attributed to cooperative conformational changes of the polypeptide chain featuring a relaxation time of about 400 ns. The distribution of the relaxation times corresponding to the second relaxation contribution was characterized by a short time cutoff, between about 0.02 and 0.4 ns depending on temperature, and a long time tail extending to microseconds. Such relaxation behavior was interpreted in terms of solute-solvent interactions reflecting various hydration layers of HSA molecules. At acid and alkaline pH, an additional Debye-type contribution with relaxation time in the range of 30-100 ns exists. It seems to be due to proton transfer reactions of protein side-chain groups. The kinetic and thermodynamic parameters of these processes have been estimated from these first measurements to indicate the potential of acoustic spectra for the investigation of the elementary kinetics of albumin processes.     

Laurdan solvatochromism: solvent dielectric relaxation and intramolecular excited-state reaction.

Absorption, steady-state, and time-resolved fluorescence measurements have been performed on laurdan dissolved either in white viscous apolar solvents or in ethanol as a function of temperature. The heterogeneity of the absorption spectra in white oils or in ethanol is consistent with semiempirical calculations performed previously on Prodan. From steady-state and time-resolved fluorescence measurements in apolar media, an excited state reaction is evidenced. The bimodal lifetime distribution determined from the maximum entropy method (MEM) analysis is attributed to the radiative deexcitation of a "locally excited" (LE) state and of a "charge transfer" (CT) state, whereas a very short component (20 ps), the sign and the amplitude of which depend on the emission wavelength, is attributed to the kinetics of the interconvertion reaction. The observation of an isoemissive point in the temperature range from -50 degrees C to -110 degrees C in ethanol suggests an interconvertion between two average excited-state populations: unrelaxed and solvent-relaxed CT states. A further decrease in temperature (-190 degrees C), leading to frozen ethanol, induces an additional and important blue shift. This low temperature spectrum is partly attributed to the radiative deexcitation of the LE state. Time-resolved emission spectra (TRES) measurements at -80 degrees C in the ethanol liquid phase show a large spectral shift of approximately 2500 cm(-1) (stabilization energy of the excited state: 7.1 kcal x M(-1)). The time-dependent fluorescence shift (TDFS) is described for its major part by a nanosecond time constant. The initial part of the spectral shift reveals, however, a subnanosecond process that can be due to fast internal solvent reorientation and/or to intramolecular excited-state reactions. These two relaxation times are also detected in the analysis of the fluorescence decays in the middle range of emission energy. The activation energy of the longest process is approximately 3 kcal x M(-1). At -190 degrees C, one subnanosecond and one nanosecond excited-state reactions are also evidenced. They are likely due to intramolecular rearrangements after the excitation, leading to the CT state and not to solvent relaxation, which is severely hindered in these temperature conditions. Therefore, both intramolecular and solvent relaxations are responsible for the large Stokes shift displayed by this probe as a function of solvent polarity. A possible scheme is proposed for the deexcitation pathway, taking into account the kinetics observed in these different solvents.     

Structural features and hydration of d(C-G-C-G-A-A-T-T-A-G-C-G); a double helix containing two G.A mispairs

Single crystal X-ray diffraction techniques have been used to characterise the molecular structure of the title compound to 2.5A resolution. The structure consists of ten standard Watson-Crick base pairs and two G.A mismatched base pairs. The purine-purine mismatches have guanine in the usual anti orientation with respect to the sugar and adenine in syn orientation. There are two hydrogen bonds formed between the mismatch bases, N-1 and O-6 of guanine with N-7 and N-6 of adenine respectively. The bulky purine-purine mismatches are accommodated with minor perturbation of the sugar-phosphate backbone. There is a slight improvement in base pair overlap at the mismatch sites. Details of the backbone conformation, base stacking interactions and hydration are presented and compared with those of the parent compound d(C-G-C-G-A-A-T-T-C-G-C-G).