Martin Volk

Determination of free energies in reaction centers of Rb. sphaeroides

Abstract Magnetic field-dependent recombination measurements together with magnetic field-dependent triplet lifetimes (Chidsey, E.D., Takiff, L., Goldstein, R.A. and Boxer, S.G. (1985) Proc. Natl. Acad. Sci USA 82, 6850–6854) yield a free energy change ΔG( P + H − − 3 P ∗) = 0.165 eV ±0.008 at 290 K. This does not depend on whether nuclear spin relaxation in the state 3P∗ is assumed to be fast or slow compared to the lifetime of this state. This value, being (almost) temperature independent, indicates ΔG( P + H − − 3 P ∗) ⋍ ΔH( P + H − − 3 P ∗) and is consistent with ΔG( 1 P ∗ − P + H − ) and ΔH( 1 P ∗ − 3 P ∗) from previous delayed fluorescence and phosphorescence data, implying ΔG ⋍ ΔH for all combinations of these states.

Multiple time scales for dispersive kinetics in early events of peptide folding

Abstract Early events involving local collapse and helix formation manifested by the disulphide recombination of a de-novo peptide, over the time scale from 1 ps to 10 μs, are shown to fit equally well to a stretched exponential ( α =0.086±0.003) and by an asymptotic power-law decay ( β =0.331±0.004). For inhomogeneous recombination kinetics each decay pattern leads to a different distribution of relaxation times.

High-Resolution Sizing of Monolayer-Protected Gold Clusters by Differential Centrifugal Sedimentation

Differential centrifugal sedimentation (DCS) has been applied to accurately size ligand-protected gold hydrosols in the 10 to 50 nm range. A simple protocol is presented to correct for particle density variations due to the presence of the ligand shell, which is formed here by either polyethylene glycol-substituted alkane thiols (PEG-alkane thiols) of different chain length or oligopeptides. The method gives reliable data for all particle sizes investigated and lends itself to rapid routine sizing of nanoparticles. Unlike TEM, DCS is highly sensitive to small changes in the thickness of the organic ligand shell and can be applied to monitor shell thickness variations of as little as 0.1 nm on particles of a given core size.

Amyloid-derived peptide forms self-assembled monolayers on gold nanoparticle with a curvature-dependent β-sheet structure.

Using a combination of Fourier transform infrared (FTIR) spectroscopy and solid-state nuclear magnetic resonance (SSNMR) techniques, the secondary structure of peptides anchored on gold nanoparticles of different sizes is investigated. The structure of the well-studied CALNN-capped nanoparticles is compared to the structure of nanoparticles capped with a new cysteine-terminated peptide, CFGAILSS. The design of that peptide is derived from the minimal amyloidogenic sequence FGAIL of the human islet polypeptide amylin. We demonstrate that CFGAILSS forms extended fibrils in solution. When constrained at a nanoparticle surface, CFGAILSS adopts a secondary structure markedly different from CALNN. Taking into account the surface selection rules, the FTIR spectra of CFGAILSS-capped gold nanoparticles indicate the formation of β-sheets which are more prominent for 25 nm diameter nanoparticles than for 5 nm nanoparticles. No intermolecular (13)C-(13)C dipolar coupling is detected with rotational resonance SSNMR for CALNN-capped nanoparticles, while CALNN is in a random coil configuration. Coupling is detected for CFGAILSS-capped gold nanoparticles, however, consistent with an intermolecular (13)C-(13)C distance of 5.0 ± 0.3 Å, in agreement with intermolecular hydrogen bonding in a parallel β-sheet structure.

Energy dissipation and relaxation processes in deoxy myoglobin after photoexcitation in the Soret region

Abstract The kinetics of the spectral evolution of myoglobin (Mb) following the Soret band (S) excitation (405 nm) were measured in the Q-band (450–630 nm) and band-III (730–900 nm) regions. There are two ultrafast electronic relaxation steps, one less than 100 fs and another of a few 100 fs that bring Mb to the ground electronic state (S0). The anisotropy reaches 0.1 by ≈400 fs and then remains constant as the system approaches equilibrium. Further relaxation involves a biphasic decay dominated by a few ps component in addition to a smaller 15 ps component. These processes are ascribed to vibrational cooling in the ground electronic state. The distinctive shapes of the transient spectra are consistent with predictions of the differences between the spectra of hot and colder S0 molecules.

Similarity of primary radical pair recombination in photosystem II and bacterial reaction centers

We report temperature and magnetic field dependent measurements of the recombination dynamics of the radical pair P680+Pheo− in D1D2cytb559 reaction centers of photosystem II and compare the results to those obtained in bacterial reaction centers. In photosystem II the rate of recombination to the groundstate is found to be slower than in the bacterial reaction centers by a factor of at least 50. This difference arises from the different redox potentials of the pigments of plant and bacterial reaction centers. In contrast, the rate of recombination to the triplet state is similar in all reaction centers, indicating a similar electronic coupling which allows us to conclude upon the structural similarity.

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 kBT). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 kBT) 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.

Fast Initiation of Peptide and Protein Folding Processes

The investigation of fast processes of peptide and protein folding has received increasing attention in the last years, driven by the development of new experimental approaches that make it possible to go beyond the millisecond time resolution of standard stopped-flow or rapid mixing techniques. The new methods allow the direct observation of important first steps such as hydrophobic collapse or secondary structure formation during the transition from the disordered polypeptide to the functional protein. However, most of these techniques are limited to a very narrow range of proteins or have other experimental restrictions and shortcomings. This review, after an overview and discussion of previously employed methods, describes a novel fast optical trigger for protein folding. This optical trigger has the potential to be used in the study of a wide variety of proteins and peptides without any of the restrictions of previous approaches. In an initial application of this technique, α-helix folding in short peptides was investigated.

Fast folding dynamics of an α-helical peptide with bulky side chains

The fast folding/unfolding dynamics of the α-helical polypeptide poly-N5-(3-hydroxypropyl)-L-glutamine, PHPG, was observed in D2O after a nanosecond laser-induced temperature jump of 1 °C. The sudden rise in temperature disturbs the helix–coil equilibrium of the peptide and the ensuing structural relaxation was monitored by time-resolved IR-spectroscopy of the amide I′ band with a time resolution of 12 ns. After an initial relaxation on the time scale of a few nanoseconds, probably arising from a local rearrangement of random coil or side chain structures, the helix–coil relaxation proceeds on the time scale of a few hundred nanoseconds. The observed time constant of 227 ns is very similar to the helix–coil relaxation time constants observed for short alanine-based peptides, in spite of the greater length and more complex and bulky side chain of PHPG.

Singlet Oxygen Generation by Laser Irradiation of Gold Nanoparticles.

The formation of singlet oxygen by irradiation of gold nanoparticles in their plasmon resonance band with continuous or pulsed laser light has been investigated. Citrate-stabilized nanoparticles were found to facilitate the photogeneration of singlet oxygen, albeit with low quantum yield. The reaction caused by pulsed laser irradiation makes use of the equilibrated hot electrons that can reach temperatures of several thousand degrees during the laser pulse. Although less efficient, continuous irradiation, which acts via the short-lived directly excited primary “hot” electrons only, can produce enough singlet oxygen for photodynamic cancer therapy and has significant advantages for practical applications. However, careful design of the nanoparticles is needed, since even a moderately thick capping layer can completely inhibit singlet oxygen formation. Moreover, the efficiency of the process also depends on the nanoparticle size.