Measurement of energy landscape roughness of folded and unfolded proteins.

The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow the motion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting from a nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in close contact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us to quantify the roughness of the energy landscape (4-5 k(B)T). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 k(B)T) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues.

Comparison of the ATP binding sites of protein kinases using conformationally diverse bisindolylmaleimides.

The conformation of a bisindolylmaleimide may be controlled by the size of a macrocyclic ring in which it is constrained. A range of techniques were used to demonstrate that the tether controls both the ratio of the two limiting conformers (syn and anti) in solution and the extent of conjugation between the maleimide and indole rings. Screening the conformationally diverse bisindolylmaleimides against a panel of protein kinases allowed their ATP binding sites to be compared using a chemical approach which, like sequence alignment, does not require detailed structural information. This approach lead to the conclusion that several AGC group protein kinases (including PKCalpha, PKCbeta, MSK1, p70 S6K, PDK-1, and MAPKAP-K1alpha) may be best inhibited by bisindolylmaleimides which adopt a compressed approximately C2-symmetric anti conformation; in constrast, GSK3beta may be best inhibited by bisindolylmaleimides whose ground state is a distorted syn conformation. It is concluded that PDK-1, whose structure has been determined by X-ray crystallography, and its mutants, may serve as particularly useful surrogates for the study of PKC inhibitors.

Synthesis and photochemistry of a new class of photocleavable protein cross-linking reagents.

A new series of photocleavable protein cross-linking reagents based on bis(maleimide) derivatives of diaryl disulfides have been synthesised. They have been functionalised with cysteine and transient absorption spectra for the photolysis reaction have been recorded by using the pump-probe technique with a time resolution of 100 femtoseconds. Photolysis of the disulfide bond yields the corresponding thiyl radicals in less than a picosecond. There is a significant amount of geminate recombination, but some of the radicals escape the solvent cage and the quantum yield for photocleavage is 30 % in water.

Energy migration in novel pH-triggered self-assembled beta-sheet ribbons.

Energy migration between tryptophan residues has been experimentally demonstrated in self-assembled peptide tapes. Each peptide contains 11 amino acids with a Trp at position 6. The peptide self-assembly is pH-sensitive and forms amphiphilic tapes, which further stack in ribbons (double tapes) and fibrils in water depending on the concentration. Fluorescence spectra, quenching, and anisotropy experiments showed that when the pH is lowered from 9 to 2, the peptide self-assembly buries the tryptophan in a hydrophobic and restricted environment in the interior of stable ribbons as expected on the basis of the peptide design. These fluorescence data support directly and for the first time the presence of such ribbons which are characterized by a highly packed and stable hydrophobic interior. In common with Trp in many proteins, fluorescence lifetimes are nonexponential, but the average lifetime is shorter at low pH, possibly due to quenching with neighboring Phe residues. Unexpectedly, time-resolved fluorescence anisotropy does not change significantly with self-assembly when in water. In highly viscous sucrose-water mixtures, the anisotropy decay at low pH was largely unchanged compared to that in water, whereas at high pH, the anisotropy decay increased significantly. We concluded that depolarization at low pH was not due to rotational diffusion but mainly due to energy migration between adjacent tryptophan residues. This was supported by a master equation kinetic model of Trp-Trp energy migration, which showed that the simulated and experimental results are in good agreement, although on average only three Trp residues were visited before emission.

Accurate analysis of fluorescence decays from single molecules in photon counting experiments.

In time-resolved, single-photon counting experiments, the standard method of nonlinear least-squares curve fitting incorrectly estimates the fluorescence lifetimes. Even for single-exponential data, errors may be up to +/- 15%, and for more complex fits, may be even higher, although the fitted line appears to describe the data. The origin of this error is not a result of the Poisson distribution, as is often stated, but is entirely due to the weighting of the fit. An alternative weighting method involving a minor change in the fitting method eliminates this problem, enabling accurate fitting even in difficult cases, including the small data sets observed in single molecule experiments and with a precision similar to that of maximum likelihood methods.

Excited state dynamics and rapid internal conversion in a stable dipole molecule.

The excited singlet state of an azomethine ylide or 'stable dipole' exhibits an ultrafast radiationless relaxation after femtosecond laser excitation. These transients are observed before the excited state decays in an almost activationless manner, the barrier is 440 cm-1, to the ground state with a 1.5 ps lifetime. Cooling of the hot ground state is also apparent in the transient absorption data and in methanol decays with a 5.7 ps lifetime. The viscosity dependence of the fluorescence yield and lifetime in different solvents is small and far less pronounced than in triphenylmethane dyes. Surprisingly, the excited state decay is not due to twisting about the C-N bond of the ylide but it is caused by buckling of one of the rings as the nitrogen atom changes character from sp2 to sp3 hybridisation.

(eta2-tetracyanoethene)bis(triphenylphosphine-kappaP)palladium-dichloromethane (1/0.7).

The title compound, [Pd(C(6)N(4))(C(18)H(15)P)(2)].0.7CH(2)Cl(2), shows a planar coordination geometry around the metal atom that is an almost perfect isosceles triangle if the tetracyanoethene (tcne) ligand is deemed to occupy a single coordination site. The framework of the tcne ligand shows small distortions due to intramolecular steric contacts between the C[triple-bond]N groups and phenyl rings of the triphenylphosphine ligands.

Femtosecond electron-transfer reactions in mono- and polynucleotides and in DNA.

Quenching of redox active, intercalating dyes by guanine bases in DNA can occur on a femtosecond time scale both in DNA and in nucleotide complexes. Notwithstanding the ultrafast rate coefficients, we find that a classical, nonadiabatic Marcus model for electron transfer explains the experimental observations, which allows us to estimate the electronic coupling (330 cm(-1)) and reorganization (8070 cm(-1)) energies involved for thionine-[poly(dG-dC)](2) complexes. Making the simplifying assumption that other charged, pi-stacked DNA intercalators also have approximately these same values, the electron-transfer rate coefficients as a function of the driving force, DeltaG, are derived for similar molecules. The rate of electron transfer is found to be independent of the speed of molecular reorientation. Electron transfer to the thionine singlet excited state from DNA obtained from calf thymus, salmon testes, and the bacterium, micrococcus luteus (lysodeikticus) containing different fractions of G-C pairs, has also been studied. Using a Monte Carlo model for electron transfer in DNA and allowing for reaction of the dye with the nearest 10 bases in the chain, the distance dependence scaling parameter, beta, is found to be 0.8 +/- 0.1 A(-1). The model also predicts the redox potential for guanine dimers, and we find this to be close to the value for isolated guanine bases. Additionally, we find that the pyrimidine bases are barriers to efficient electron transfer within the superexchange limit, and we also infer from this model that the electrons do not cross between strands on the picosecond time scale; that is, the electronic coupling occurs predominantly through the pi-stack and is not increased substantially by the presence of hydrogen bonding within the duplex. We conclude that long-range electron transfer in DNA is not exceptionally fast as would be expected if DNA behaved as a "molecular wire" but nor is it as slow as is seen in proteins, which do not benefit from pi-stacking.