Studying excited states of proteins by NMR spectroscopy.

Protein structure is inherently dynamic, with function often predicated on excursions from low to higher energy conformations. For example, X-ray studies of a cavity mutant of T4 lysozyme, L99A, show that the cavity is sterically inaccessible to ligand, yet the protein is able to bind substituted benzenes rapidly. We have used novel relaxation dispersion NMR techniques to kinetically and thermodynamically characterize a transition between a highly populated (97%, 25 degrees C) ground state conformation and an excited state that is 2.0 kcal mol(-1) higher in free energy. A temperature-dependent study of the rates of interconversion between ground and excited states allows the separation of the free energy change into enthalpic (Delta H = 7.1 kcal mol(-1)) and entropic (T Delta S = 5.1 kcal mol(-1), 25 degrees C) components. The residues involved cluster about the cavity, providing evidence that the excited state facilitates ligand entry.     

Temperature and pressure denaturation of chignolin: Folding and unfolding simulation by multibaric-multithermal molecular dynamics method

A multibaric‐multithermal molecular dynamics (MD) simulation of a 10‐residue protein, chignolin, was performed. All‐atom model with the Amber parm99SB force field was used for the protein and the TIP3P model was used for the explicit water molecules. This MD simulation covered wide ranges of temperature between 260 and 560 K and pressure between 0.1 and 600 MPa and sampled many conformations without getting trapped in local‐minimum free‐energy states. Folding events to the native β‐hairpin structure occurred five times and unfolding events were observed four times. As the temperature and/or pressure increases, fraction of folded chignolin decreases. The partial molar enthalpy change ΔH and partial molar volume change ΔV of unfolding were calculated as ΔH = 24.1 ± 4.9 kJ/mol and ΔV = ?5.6 ± 1.5 cm³/mol, respectively. These values agree well with recent experimental results. Illustrating typical local‐minimum free‐energy conformations, folding and unfolding pathways were revealed. When chignolin unfolds from the β‐hairpin structure, only the C terminus or both C and N termini open first. It may undergo an α‐helix or 3₁₀‐helix structure and finally unfolds to the extended structure. Difference of the mechanism between temperature denaturation and pressure denaturation is also discussed. Temperature denaturation is caused by making the protein transferred to a higher entropy state and making it move around more with larger space. The reason for pressure denaturation is that water molecules approach the hydrophobic residues, which are not well hydrated at the folded state, and some hydrophobic contacts are broken. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Cavity as a Source of Conformational Fluctuation and High-Energy State: High-Pressure NMR Study of a Cavity-Enlarged Mutant of T4Lysozyme

Although the structure, function, conformational dynamics, and controlled thermodynamics of proteins are manifested by their corresponding amino acid sequences, the natural rules for molecular design and their corresponding interplay remain obscure. In this study, we focused on the role of internal cavities of proteins in conformational dynamics. We investigated the pressure-induced responses from the cavity-enlarged L99A mutant of T4 lysozyme, using high-pressure NMR spectroscopy. The signal intensities of the methyl groups in the ¹H/¹³C heteronuclear single quantum correlation spectra, particularly those around the enlarged cavity, decreased with the increasing pressure, and disappeared at 200 MPa, without the appearance of new resonances, thus indicating the presence of heterogeneous conformations around the cavity within the ground state ensemble. Above 200 MPa, the signal intensities of >20 methyl groups gradually decreased with the increasing pressure, without the appearance of new resonances. Interestingly, these residues closely matched those sensing a large conformational change between the ground- and high-energy states, at atmospheric pressure. ¹³C and ¹H NMR line-shape simulations showed that the pressure-induced loss in the peak intensity could be explained by the increase in the high-energy state population. In this high-energy state, the aromatic side chain of F114 gets flipped into the enlarged cavity. The accommodation of the phenylalanine ring into the efficiently packed cavity may decrease the partial molar volume of the high-energy state, relative to the ground state. We suggest that the enlarged cavity is involved in the conformational transition to high-energy states and in the volume fluctuation of the ground state.     

Molecular dynamics study of early picosecond events in the bacteriorhodopsin photocycle: dielectric response, vibrational cooling and the J, K intermediates.

Molecular dynamics simulations have been carried out to study the J625 and K590 intermediates of bacteriorhodopsin's (bRs) photocycle starting from a refined structure of bR568. The coupling between the electronic states of retinal and the protein matrix is characterized by the energy difference delta E(t) between the excited state and the ground state to which the protein contributes through the Coulomb interaction. Our simulations indicate that the J625 intermediate is related to a polarization of the protein matrix due to the brief (200 fs) change of retinal's charge distribution in going to the excited state and back to the ground state, and that the rise time of the K590 intermediate is determined by vibrational cooling of retinal.     

A heteropolymer model study for the mechanism of protein folding

A heteropolymer model of randomly self-interacting chains in two dimensions is studied with numerical simulations in order to elucidate the folding mechanism of protein. We find that the model occasionally shows folding propensity depending on the sequence of random numbers given to the chain. We study the thermodynamic and kinematic roles in the folding mechanism by grouping the local energy minima found in the simulations into clusters according to the similarity of their conformations. It is suggested that the local minima to which some heteropolymers show a folding tendency are always the lowest energy states of the energy spectrum within a cluster, though which cluster is selected depends on the sequence. For the eight random sequences we study, we find that the energy gap between the ground state and excited states is little correlated with folding or nonfolding. We rather find that folding propensities are correlated with the global structure of the average energy surface, implying a dominant kinetic role in the folding mechanism, although thermal factors cannot be ignored as the mechanism of choosing the ground state within a cluster of states connected by small deformations. We suggest that a hierarchical cluster structure plays an important role in selecting a unique folded state out of the huge number of local minima of heteropolymers. © 1997 John Wiley & Sons, Inc.     

Studying pressure denaturation of a protein by molecular dynamics simulations

Many globular proteins unfold when subjected to several kilobars of hydrostatic pressure. This “unfolding‐up‐on‐squeezing” is counter‐intuitive in that one expects mechanical compression of proteins with increasing pressure. Molecular simulations have the potential to provide fundamental understanding of pressure effects on proteins. However, the slow kinetics of unfolding, especially at high pressures, eliminates the possibility of its direct observation by molecular dynamics (MD) simulations. Motivated by experimental results—that pressure denatured states are water‐swollen, and theoretical results—that water transfer into hydrophobic contacts becomes favorable with increasing pressure, we employ a water insertion method to generate unfolded states of the protein Staphylococcal Nuclease (Snase). Structural characteristics of these unfolded states—their water‐swollen nature, retention of secondary structure, and overall compactness—mimic those observed in experiments. Using conformations of folded and unfolded states, we calculate their partial molar volumes in MD simulations and estimate the pressure‐dependent free energy of unfolding. The volume of unfolding of Snase is negative (approximately ?60 mL/mol at 1 bar) and is relatively insensitive to pressure, leading to its unfolding in the pressure range of 1500–2000 bars. Interestingly, once the protein is sufficiently water swollen, the partial molar volume of the protein appears to be insensitive to further conformational expansion or unfolding. Specifically, water‐swollen structures with relatively low radii of gyration have partial molar volume that are similar to that of significantly more unfolded states. We find that the compressibility change on unfolding is negligible, consistent with experiments. We also analyze hydration shell fluctuations to comment on the hydration contributions to protein compressibility. Our study demonstrates the utility of molecular simulations in estimating volumetric properties and pressure stability of proteins, and can be potentially extended for applications to protein complexes and assemblies. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Protein folding in the absence of chemical denaturants. Reversible pressure denaturation of the noncovalent complex formed by the association of two protein fragments

Small monomeric proteins are the best models for studying protein folding, but they are often too stable for denaturation using pressure as the sole perturbant. In the present work we subject [CI-2(1-40).(41-64)], a noncovalent complex formed by the association of two complementary fragments of the chymotrypsin inhibitor-2, to high pressure to investigate the folding mechanism of a model protein. Pressures up to 3.5 kilobar do not affect the intact protein, but it can be unfolded reversibly by pressure in the presence of subdenaturing concentrations of guanidine chloride, with free energy and molar volume changes of 2.5 kcal mol-1 and 42.5 ml mol-1, respectively. In contrast, the complex can be reversibly denatured by high pressure without the addition of chemical denaturants. However, the process is clearly independent of the protein concentration, indicating lack of dissociation. We determined a change in the free energy of 1.4 kcal mol-1 and a molar volume change of 35 ml mol-1 for the pressure denaturation of the complex. A persistent quenching of the tryptophan adds further evidence for the presence of residual structure in the high pressure-denatured state. This state also appears to be compact as the small volume change indicates, compared with pressure denaturation of naturally occurring dimers. Based on observations of a number of pressure-denatured states and on characteristics of large CI-2 fragments with a solvent accessible core but maintaining tertiary interactions, the structure of the pressure-denatured state of the CI-2 complex could be explained by an ordered molten globule-like conformation.     

Observations concerning light promoted electronic delocalization in covalently linked transition metal complexes

The emission spectral band shapes of several polypyridine-ligand (PP) bridged bis-ruthenium(II) complexes imply that Ru(II)/Ru(III) electronic coupling is weaker in their lowest energy metal to ligand charge transfer (MLCT) excited states than in their corresponding mixed valence ground states. In general, the amplitudes of the vibronic contributions to emission band shapes decrease markedly with the excited state-ground state energy differences, and it is expected that complexes with degenerate, or mixed valence excited states will have very weak vibronic side bands if configurational mixing of the degenerate MLCT excited states is substantial. However, the bimetallic PP-bridged ruthenium complexes emit at significantly lower energy than their monometallic analogs, but the vibronic contributions to their 77 K emission spectra are very similar to those of their monometallic complexes analogs. This indicates that the mixed valence excited states of the bimetallic complexes are electronically localized.     

Stability of subtilisin and lysozyme under high hydrostatic pressure

The stabilities of subtilisin and lysozyme under hydrostatic pressures up to 200 MPa were investigated for up to 7 days at 25 degrees C. Methods were chosen to assess changes in tertiary and secondary protein structure as well as aggregation state. Tertiary structure was monitored in situ with second derivative UV spectroscopy and after pressure treatment by dynamic light scattering and second derivative UV spectroscopy. Secondary structure and potential secondary structural changes were characterized by second derivative FTIR spectroscopy. Changes in aggregation state were assessed using dynamic light scattering. Additionally, protein concentration balances were carried out to detect any loss of protein as a function of pressure. For the conditions tested, neither protein shows measurable changes in tertiary or secondary structure or signs of aggregation. Lysozyme concentration balances show no dependence on pressure. Subtilisin concentration balances at high protein concentration (4 mg/mL and higher) do not show pressure dependence. However, the concentration balances carried out at 0.4 mg/mL show a clear sign of pressure dependence. These results may be explained by protein interaction with the vial surface and appear to be rate limited by the equilibrium between active and inactive protein on the surface. Pressure increases protein loss, and the estimated partial molar volume change between the two states is estimated to be -20 +/- 10 mL/mol.     

Probing slowly exchanging protein systems via 13Cα-CEST: monitoring folding of the Im7 protein

A ¹³C^α chemical exchange saturation transfer based experiment is presented for the study of protein systems undergoing slow interconversion between an ‘observable’ ground state and one or more ‘invisible’ excited states. Here a labeling strategy whereby [2-¹³C]-glucose is the sole carbon source is exploited, producing proteins with ¹³C at the C^α position, while the majority of residues remain unlabeled at CO or C^β. The new experiment is demonstrated with an application to the folding reaction of the Im7 protein that involves an on-pathway excited state. The obtained excited state ¹³C^α chemical shifts are cross validated by comparison to values extracted from analysis of CPMG relaxation dispersion profiles, establishing the utility of the methodology.     

Low-lying electronic states of the ferrous high-spin (S=2) heme in deoxy-Mb and deoxy-Hb studied by highly-sensitive multi-frequency EPR

The low-lying electronic states of the ferrous high-spin heme in deoxy-myoglobin (deoxy-Mb) and deoxy-hemoglobin (deoxy-Hb) were probed by multi-frequency electron paramagnetic resonance (MFEPR) spectroscopy. An unexpected broad EPR signal was measured at the zero magnetic field using cavity resonators at 34-122 GHz that could not be simulated using any parameter sets for the S = 2 spin Hamiltonian assuming spin quintet states in the ⁵B₂ ground state. Furthermore, we have observed novel, broad EPR signals measured at 70-220 GHz and 1.5 K using a single pass transmission probe. These signals are attributed to the ferrous high-spin heme in deoxy-Mb and deoxy-Hb. The resonant peaks shifted to a higher magnetic field with increasing frequency. The energy level separation between the ground singlet and the first excited state at the zero magnetic field was directly estimated to be 3.5 cm^− 1 for deoxy-Hb. For deoxy-Mb, the first two excited singlet states are separated by 3.3 cm^− 1 and 6.5 cm^− 1, respectively, from the ground state. The energy gap at the zero magnetic field is directly derived from our MFEPR for deoxy-Mb and deoxy-Hb and strongly supports the theoretical analyses based on the Mössbauer and magnetic circular dichroism experiments.     

Selected-fit versus induced-fit protein binding: kinetic differences and mutational analysis

The binding of a ligand molecule to a protein is often accompanied by conformational changes of the protein. A central question is whether the ligand induces the conformational change (induced-fit), or rather selects and stabilizes a complementary conformation from a pre-existing equilibrium of ground and excited states of the protein (selected-fit). We consider here the binding kinetics in a simple four-state model of ligand-protein binding. In this model, the protein has two conformations, which can both bind the ligand. The first conformation is the ground state of the protein when the ligand is off, and the second conformation is the ground state when the ligand is bound. The induced-fit mechanism corresponds to ligand binding in the unbound ground state, and the selected-fit mechanism to ligand binding in the excited state. We find a simple, characteristic difference between the on- and off-rates in the two mechanisms if the conformational relaxation into the ground states is fast. In the case of selected-fit binding, the on-rate depends on the conformational equilibrium constant, whereas the off-rate is independent. In the case of induced-fit binding, in contrast, the off-rate depends on the conformational equilibrium, while the on-rate is independent. Whether a protein binds a ligand via selected-fit or induced-fit thus may be revealed by mutations far from the protein's binding pocket, or other "perturbations" that only affect the conformational equilibrium. In the case of selected-fit, such mutations will only change the on-rate, and in the case of induced-fit, only the off-rate.     

On the photophysics of metallophthalocyanine-based photothermal sensitizers: synergism between theory and experiment

A comprehensive understanding of the factors governing the efficiency of metallophthalocyanine-based photothermal sensitizers requires the knowledge of their excited-state dynamics. This can only be properly gained when the nature and energy of the excited states (often spectroscopically silent) lying between the photogenerated state and the ground state are known. Here the excited state deactivation mechanism of two very promising metallophthalocyanine-based photothermal sensitizers, NiPc(OBu)(8) and NiNc(OBu)(8), is reviewed. It is shown that time dependent density functional theory (TDDFT) methods are capable to provide reliable information on the nature and energies of the low-lying excited states along the relaxation pathways. TDDFT calculations and ultrafast experiments consistently show that benzoannulation of the Pc ring modifies the photodeactivation mechanism of the photogenerated S(1)(pi,pi*) state by inducing substantial changes in the relative energies of the excited states lying between the S(1)(pi,pi*) state and the ground state.     

Phototautomerism of the GC base pair of DNA

Electronic spectra and ground and excited state electronic structures of normal G and rare tautomeric G1z.sbnd;C1 base pairs as well as of the individual rare tautomeric bases (purines and pyrimidines) have been studied using the VE-PPP molecular orbital method. The nature and consequences of the lowest energy purine-localized and purine to pyrimidine charge transfer type π?π1 singlet excitations of the base pairs have been investigated. The results indicate that in these excited states, particularly in the charge transfer excited state, the probability for the GC base pair to change over to G1C1 would be larger than in the ground state. The likeliness of the relevance of results obtained experimentally by other workers from the study of a model system to the GC base pair is discussed.     

The photophysics of Ni(II) meso-tetraphenylbenzoporphyrin: A combined theoretical and experimental investigation

A combined theoretical and kinetic spectrometric investigation of Ni(II)-tetraphenyl tetrabenzoporphyrin (NiTPTBP) has demonstrated that photo-excitation into the B band (400 nm) of the ground state absorption spectrum rapidly generates the compound in its Q state. The intriguing time dependence of stimulated emission generated from the Q state implies that this state is populated via two (or more) radiationless channels, one is very rapid (<1 ps) and another less rapid (∼6 ps). The theoretical results show that two excited states with significant oscillator strengths lie close in energy within the B band envelope. These are the 5 ¹E (2.72 eV; f = 0.95) and the 4 ¹E (2.63 eV; f = 0.19) and both these could be directly excited by the 400 nm excitation pulse, in which case these two upper states could be the origins of the two kinetic populations of the Q state. Less than 2 ps post excitation onset the spectral characteristics of a hot metal-centered excited state appear, identified as 2 ¹E. The excess vibrational energy of this state is dissipated by IVR and intermolecular modes. Eventually the cooled (d,d) state repopulates the initial ground state with a rate that is extremely solvent dependent. The 2 ¹E state is computed to possess some 20% of MLCT character, which is the possible source of the solvent dependent deactivation rate.     

Resolving the excited state equilibrium of peridinin in solution

The carotenoid peridinin is abundant in the biosphere, as it is the main pigment bound by the light-harvesting complexes of dinoflagellates, where it collects blue and green sunlight and transfers energy to chlorophyll a with high efficiency. Its molecular structure is particularly complex, giving rise to an intricate excited state manifold, which includes a state with charge-transfer character. To disentangle the excited states of peridinin and understand their function in vivo, we applied dispersed pump-probe and pump-dump-probe spectroscopy. The preferential depletion of population from the intramolecular charge transfer state by the dump pulse demonstrates that the S(1) and this charge transfer state are distinct entities. The ensuing dump-induced dynamics illustrates the equilibration of the two states which occurs on the time scale of a few picoseconds. Additionally, the dump pulse populates a short-lived ground state intermediate, which is suggestive of a complex relaxation pathway, probably including structural reorientation or solvation of the ground state. These findings indicate that the unique intramolecular charge transfer state of peridinin is an efficient energy donor to chlorophyll a in the peridinin-chlorophyll-protein complex and thus plays a significant role in global light harvesting.     

Structures of polypyridine mononuclear Ir complexes in the ground state and the lowest triplet state

The structures of eight Ir^III centered polypyridine complexes were determined by density-functional-theory calculations. The differences in the optimized geometries between the ground state and the lowest excited triplet state were mainly considered. A crystal structure of [IrCl(bpy)(terpy)](PF₆)₂ was also obtained by the X-ray diffraction study, where bpy is 2,2′-bipyridine and terpy is 2,2′:6′,2″-terpyridine. The computed geometries are in good agreement with the experimental ones. Those in the triplet biradical states were determined to evaluate the energy difference between the triplet and the ground states. The resulted values correlate well with the observed emission energies. To investigate the nature of the electronic transition involving the ground and the first excited triplet states, a Mulliken population analysis of the spin densities on the eight complexes was performed. The geometric changes from free tterpy ligand {tterpy = 4′-(4-tolyl)-2,2′:6′,2″-terpyridine} to the Ir^III complexed ligand, and then to triplet biradical were examined. The planarity enhanced the π-π^∗ excitation in the ligand and consequently gave the stable triplet biradical of the complex. It was found that efficient phosphorescence should be impacted by the presence of one coplanar polypyridine ligand.     

Computational (DFT and TD DFT) study of the electron structure of the tautomers/conformers of uridine and deoxyuridine and the processes of intramolecular proton transfers

Six uridine and six deoxyuridine isomers were studied at the B3LYP and TD B3LYP theoretical level and 6–31+G(d) basis function. The stability and the excited states of the isomers were studied in order to clarify some known experimental data. It was established that the rotation of the oxo uracil ring in uridine is energetically more likely to occur in the excited state than in the ground state, driven by the bright ¹ ππ* state and the dark charge transfer ¹nπ* state. Very high energy barriers (on the S_o) were found for thermal intramolecular proton transfer processes.     

Comparative study of the relaxation mechanisms of the excited states of cytosine and isocytosine

An experimental and theoretical investigation was performed to study the photostability of cytosine and isocytosine. The experimental UV irradiation of acetonitrile solutions of the two compounds showed that the amino-oxo tautomer of cytosine is photostable while the amino-oxo tautomer of isocytosine tautomerizes to the amino-hydroxy form. The theoretical investigations were carried out at the CC2 level of theory. They were performed to explain the experimental observations. It was found that the ¹ππ^* excited states of the ring deformation mechanisms of cytosine and isocytosine relax (internal conversion) to the ground states of the amino-oxo forms of the compounds. We propose a channel for the radiationless deactivation of the repulsive ¹πσ^* excited state of the amino-oxo form of isocytosine to the ground state of the amino-hydroxy tautomer.