A switch from α-helical to ß-strand conformation during co-translational protein folding.

Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo-EM structure determination to show that folding of a ß-barrel protein begins with formation of a dynamic α-helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N-terminal part of the nascent chain refolds to a ß-hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α-helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl-transferase center suggest that protein folding could modulate ribosome activity.

Lateral gate dynamics of the bacterial translocon during cotranslational membrane protein insertion.

During synthesis of membrane proteins, transmembrane segments (TMs) of nascent proteins emerging from the ribosome are inserted into the central pore of the translocon (SecYEG in bacteria) and access the phospholipid bilayer through the open lateral gate formed of two helices of SecY. Here we use single-molecule fluorescence resonance energy transfer to monitor lateral-gate fluctuations in SecYEG embedded in nanodiscs containing native membrane phospholipids. We find the lateral gate to be highly dynamic, sampling the whole range of conformations between open and closed even in the absence of ligands, and we suggest a statistical model-free approach to evaluate the ensemble dynamics. Lateral gate fluctuations take place on both short (submillisecond) and long (subsecond) timescales. Ribosome binding and TM insertion do not halt fluctuations but tend to increase sampling of the open state. When YidC, a constituent of the holotranslocon, is bound to SecYEG, TM insertion facilitates substantial opening of the gate, which may aid in the folding of YidC-dependent polytopic membrane proteins. Mutations in lateral gate residues showing in vivo phenotypes change the range of favored states, underscoring the biological significance of lateral gate fluctuations. The results suggest how rapid fluctuations of the lateral gate contribute to the biogenesis of inner-membrane proteins.

Geminate charge recombination in polymer/fullerene bulk heterojunction films and implications for solar cell function.

We have studied the influence of three different fullerene derivatives on the charge generation and recombination dynamics of polymer/fullerene bulk heterojunction (BHJ) solar cell blends. Charge generation in APFO3/[70]PCBM and APFO3/[60]PCBM is very similar and somewhat slower than charge generation in APFO3/[70]BTPF. This difference qualitatively matches the trend in free energy change of electron transfer estimated from the LUMO energies of the polymer and fullerene derivatives. The first order (geminate) charge recombination rate is significantly different for the three fullerene derivatives studied and increases in the order APFO3/[70]PCBM < APFO3/[60]PCBM < APFO3/[70]BTPF. The variation in electron transfer rate cannot be explained from the LUMO energies of the fullerene derivatives and single-step electron transfer in the Marcus inverted region and simple considerations of expected trends for the reorganization energy and free energy change. Instead we suggest that geminate charge recombination occurs from a state where electrons and holes have separated to different distances in the various materials because of an initially high charge mobility, different for different materials. In a BHJ thin film this charge separation distance is not sufficient to overcome the electrostatic attraction between electrons and holes and geminate recombination occurs on the nanosecond to hundreds of nanoseconds time scale. In a BHJ solar cell, we suggest that the internal electric field in combination with polarization effects and the dynamic nature of polarons are key features to overcome electron-hole interactions to form free extractable charges.

Photophysics of self-assembled zinc porphyrin-bidentate diamine ligand complexes.

The effects of complexation--by bidentate nitrogen-containing ligands such as pyrazine and 4,4'-bipyridine commonly used for porphyrin self-assembly--on the photophysics of the model metalloporphyrin, ZnTPP, are reported. Ligation to form the 5-coordinate species introduces an intramolecular charge transfer (ITC) state that, depending on the oxidation and reduction potentials of the electron donor and acceptor, can become involved in the excited state relaxation processes. For ZnTPP, ligation with pyridine has little effect on excited state relaxation following either Q-band or Soret band excitation. However, coordination of ZnTPP with pyrazine and bipyridine causes the S(2) (Soret) state of the ligated species to decay almost exclusively via an S(2)-ICT-S(1) pathway, while affecting the S(1) decay route only slightly. In these 5-coordinate species the S(2)-ICT-S(1) decay route is ultrafast and nearly quantitative. Literature redox data for other bidentate ligands such as DABCO and multidentate ligands commonly used for pophyrin assembly suggest that the ITC states introduced by them could also modify the excited state relaxation dynamics of a wide variety of multiporphyrin arrays.

Gradual compaction of the nascent peptide during cotranslational folding on the ribosome.

Nascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force-profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the µs time scale. The fluctuations slow down as the domain moves away from the ribosome. Mutations that destabilize the packing of the domain's hydrophobic core have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.

Photophysics of soret-excited tetrapyrroles in solution. IV. Radiationless decay and triplet-triplet annihilation investigated using tetraphenylporphinato Sn(IV).

The S(2) population decay rates and triplet-triplet annihilation efficiencies of Sn(IV)Cl(2)TPP have been measured in fluid solutions using its weak S(2)-S(0) fluorescence as a metric. A detailed description of the excited-state photophysics of Sn(IV)Cl(2)TPP has allowed comparisons to be made between this rigid, D(4h) axially coordinated molecule and axially uncoordinated tetrapyrroles of greater flexibility and lower symmetry. S(2)-S(1) internal conversion is the major S(2) population decay path for Sn(IV)Cl(2)TPP as it is for the S(2) states of all other d(0) and d(10) metalated tetrapyrroles. The S(2) state of Sn(IV)Cl(2)TPP exhibits S(2)-S(1) relaxation rates that follow the energy gap law of radiationless transition theory and are only slightly faster than those exhibited by MgTPP and the weak coupling limit. Differences in S(2)-S(1) radiationless decay rates among the series MTPP (M = Mg, Zn, Cd, SnCl(2)) cannot be traced to differences in the displacements of the S(2) and S(1) potential surfaces. Instead, the most likely source of the large differences in S(2)-S(1) radiationless decay rates between CdTPP and Sn(IV)Cl(2)TPP is the lower symmetry of the former (near C(4v)), which permits a much larger number of vibrations to participate in S(2)-S(1) vibronic coupling. Triplet-triplet annihilation of the type 2T(1) --> S(2) + S(0) has been observed in Sn(IV)Cl(2)TPP for the first time, but is of substantially lower efficiency than seen in ZnTPP in noncoordinating solvents because of its shorter triplet lifetime and the shielding effects of its axial Cl ligands, which tend to block the short-range interaction needed for Dexter energy transfer.

Imaging techniques. Ultrafast low-energy electron diffraction in transmission resolves polymer/graphene superstructure dynamics.

Two-dimensional systems such as surfaces and molecular monolayers exhibit a multitude of intriguing phases and complex transitions. Ultrafast structural probing of such systems offers direct time-domain information on internal interactions and couplings to a substrate or bulk support. We have developed ultrafast low-energy electron diffraction and investigate in transmission the structural relaxation in a polymer/graphene bilayer system excited out of equilibrium. The laser-pump/electron-probe scheme resolves the ultrafast melting of a polymer superstructure consisting of folded-chain crystals registered to a free-standing graphene substrate. We extract the time scales of energy transfer across the bilayer interface, the loss of superstructure order, and the appearance of an amorphous phase with short-range correlations. The high surface sensitivity makes this experimental approach suitable for numerous problems in ultrafast surface science.

Chemical vapor deposition of graphene on a "peeled-off" epitaxial Cu(111) foil: a simple approach to improved properties.

We present a simple approach to improving the quality of CVD grown graphene, exploiting a Cu(111) foil catalyst. The catalyst is epitaxially grown by evaporation on a single crystal sapphire substrate, thickened by electroplating, and peeled off. The exposed surface is atomically flat, easily reduced, and exclusively of (111) orientation. Graphene grown on this catalyst under atmospheric CVD conditions and without wet chemical prereduction produces single crystal domain sizes of several hundred micrometers in samples that are many centimeters in size. The graphene produced in this way can easily be transferred to other substrates using well-established techniques. We report mobilities extracted using field-effect (as high as 29 000 cm(2) V(-1) s(-1)) and Hall bar measurement (up to 10 100 cm(2) V(-1) s(-1)).

Geminate charge recombination in alternating polyfluorene copolymer/fullerene blends.

By measuring excited state and charge dynamics in blends of an alternating polyfluorene copolymer and fullerene derivative over nine orders in time and two orders in light intensity, we have monitored the light-induced processes from ultrafast charge photogeneration to much slower decay of charges by recombination. We find that at low light intensities relevant to solar cell operation relatively fast (approximately 30 ns) geminate recombination is the dominating charge decay process, while nongeminate recombination has a negligible contribution. The conclusion of our work is that under solar illumination conditions geminate recombination of charges may be directly competing with efficient charge collection in polymer/fullerene solar cells.

Intramolecular fluorescence quenching in luminescent copolymers containing fluorenone and fluorene units: a direct measurement of intrachain exciton hopping rate.

The quenching process of fluorescence emission in polyfluorene (PF) due to the presence of intramolecular 9-fluorenone (9 FL) moieties is studied in dilute toluene solution as a function of 9 FL content in eight copolymers containing both fluorene and fluorenone units (PF/FL(x)). The absorption spectrum of PF/FL(x) copolymers clearly shows a new absorption band, redshifted relatively to the PF and 9-fluorenone absorption, which increases in intensity when the fluorenone fraction increases and also decreases with solvent polarity. Fluorescence emission spectra of PF/FL(x) show that this redshifted and unstructured emission does not coincide with the 9-fluorenone emission and, with increasing solvent polarity, it further redshifts and decreases in intensity. An isoemissive point is clearly observed on the fluorescence emission spectra of PF/FL(x) as a function of fluorenone content, showing that the new emission band is formed at the expense of PF. We propose the formation of an intramolecular charge transfer complex (ICTC) between PF units and 9-fluorenone to explain the appearance of the new emission band. Global analysis of time resolved fluorescence decays collected at 415 nm (PF emission) and 580 nm (the ICTC emission) show that three exponentials are generally needed to achieve excellent fits. Two of the components (420 ps and 6.5 ns) are independent of 9-fluorenone fraction. A further fast component is strongly dependent on fluorenone fraction and ranges between 280 and 70 ps. This component appears as a decay time at 415 nm and as a rise time at 580 nm and is ascribed to the migration of exciton to quenching sites (formation of intramolecular CT complex or exciton ionization at CT complex). A kinetic mechanism involving three different kinetic species, quenched PF units kinetically coupled with the ICTC complex, and unquenched PF units is proposed to explain the experimental data and the quenching rate constant is obtained, k(1) congruent with 10(11) s(-1). This is an experimental measurement of the intrachain exciton hopping rate.