Photochemically induced electron transfer

Biochemical reactions involving electron transfer between substrates or enzyme cofactors are both common and physiologically important; they have been studied by means of a variety of techniques. In this paper we review the application of photochemical methods to the study of intramolecular electron transfer in hemoproteins, thus selecting a small, well-defined sector of this otherwise enormous field. Photoexcitation of the heme populates short-lived excited states which decay by thermal conversion and do not usually transfer electrons, even when a suitable electron acceptor is readily available, e.g., in the form of a second oxidized heme group in the same protein; because of this, the experimental setup demands some manipulation of the hemoprotein. In this paper we review three approaches that have been studied in detail: (i) the covalent conjugation to the protein moiety of an organic ruthenium complex, which serves as the photoexcitable electron donor (in this case the heme acts as the electron acceptor); (ii) the replacement of the heme group with a phosphorescent metal-substituted porphyrin, which on photoexcitation populates long-lived excited states, capable of acting as electron donors (clearly the protein must contain some other cofactor acting as the electron acceptor, most often a second heme group in the oxidized state); (iii) the combination of the reduced heme with CO (the photochemical breakdown of the iron-CO bond yields transiently the ground-state reduced heme which is able to transfer one electron (or a fraction of it) to an oxidized electron acceptor in the protein; this method uses a "mixed-valence hybrid" state of the redox active hemoprotein and has the great advantage of populating on photoexcitation an electron donor at physiological redox potential).     

Differential native state ruggedness of the two Ca2+-binding domains in a Ca2+ sensor protein

Characterization of near-native excited states of a protein provides insights into various biological functions such as co-operativity, protein-ligand, and protein-protein interactions. In the present study, we investigated the ruggedness of the native state of EhCaBP using nonlinear temperature dependence of backbone amide-proton chemical shifts. EhCaBP is a two-domain EF-hand calcium sensor protein consisting of two EF-hands in each domain and binds four Ca2+ ions. It has been observed that approximately 30% of the residues in the protein access alternative conformations. Theoretical modeling suggested that these low-energy excited states are within 2-3 kcal/mol from the native state. Further, it is interesting to note that the residues accessing alternative conformations are more dominated in the C-terminal domain compared with its N-terminal counterpart suggesting that the former is more rugged in its native state. These distinct characteristics of N- and C-terminal domains of a calcium sensor protein belonging to the super family of calmodulin would have implications for domain dependent Ca2+ signaling pathways.     

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.     

Electronic excited states and excitation transfer kinetics in the Fenna-Matthews-Olson protein of the photosynthetic bacterium Prosthecochloris aestuarii at low temperatures

The molecular structure-function relationship of the Fenna-Matthews-Olson light-harvesting complex of the photosynthetic green bacterium Prosthecochloris aestuarii has been investigated. It has been assumed that the electronic excited states responsible for the function (transfer of electronic excitation energy) result from the dipole-dipole interactions between the bacteriochlorophyll molecules bound to the polypeptide chain of the complex at a specific three-dimensional geometry. The molecular structure-electronic excited states relationship has been addressed on the basis of simultaneous simulations of several spectroscopic observations. Current electronic excited state models for the Fenna-Matthews-Olson complex have generally been based on obtaining an optimal match between the information contents of the optical steady-state spectra and the bacteriochlorophyll organization. Recent kinetic and spectral information gathered from ultrafast time-resolved measurements have not yet been used effectively for further refinement of the excited state models and for quantification of the relation between the excited states and the energy transfer processes. In this study, we have searched for a model that not only can explain the key features of several steady-state spectra but also the temporal and spectral evolution observed in a recent absorption difference experiment and we have discussed the implications of this model for equilibration of the electronic excitation energy in systems at low temperatures. Received: 12 June 1998 / Revised version: 19 October 1998 / Accepted: 30 November 1998     

A novel mechanism for the modulation of the Ras-effector interaction by small molecules

When proteins require different conformations for their biological function, all these functional states have to coexist simultaneously in solution. However, the corresponding Gibbs free energy differences are usually rather high and thus the conformation with lowest energy predominates in solution whereas the populations of the states with higher energy (excited states) are very small. A stabilization of these excited states can be used as a novel principle to influence the activity of proteins by small molecules. For a proof of this principle, we selected the Ras protein that was shown by (31)P NMR spectroscopy to exist in solution in at least two different conformational states in its GTP form. One of these states shows a drastically reduced affinity to effectors. With Zn(2+)-cyclen we found a small molecule which selectively stabilizes the weak-binding state. It may serve as lead compound for the development of a new type of Ras-inhibitors.     

Design of protein conformational switches

Protein conformational switches are ubiquitous in nature and often regulate key biological processes. To design new proteins that can switch conformation, protein designers have focused on the two key components of protein switches: the amino acid sequence must be compatible with the multiple target states and there must be a mechanism for perturbing the relative stability of these states. Proteins have been designed that can switch between folded and disordered states, between distinct folded states and between different aggregation states. A variety of trigger mechanisms have been used, including pH shifts, post-translational modification and ligand binding. Recently, computational protein design methods have been applied to switch design. These include algorithms for designing novel ligand-binding sites and simultaneously optimizing a sequence for multiple target structures.     

Excited state properties of lanthanide complexes: Beyond ff states

Generally, metal-centered ff states dominate the discussion of the excited state properties of lanthanide complexes. In particular, the luminescence properties of Eu(III) and Tb(III) compounds have been studied in great detail for many decades. However, other types of excited states such as MC fd, MLCT, LMCT, MMCT and IL are also of interest. In this context, we have recently examined the excited state behavior of selected Ce(III), Ce(IV), Eu(II) and Gd(III) complexes which are luminescent and/or photoreactive.     

An improved ultrafast 2D NMR experiment: Towards atom-resolved real-time studies of protein kinetics at multi-Hz rates

Multidimensional NMR spectroscopy is a well-established technique for the characterization of structure and fast-time-scale dynamics of highly populated ground states of biological macromolecules. The investigation of short-lived excited states that are important for molecular folding, misfolding and function, however, remains a challenge for modern biomolecular NMR techniques. Off-equilibrium real-time kinetic NMR methods allow direct observation of conformational or chemical changes by following peak positions and intensities in a series of spectra recorded during a kinetic event. Because standard multidimensional NMR methods required to yield sufficient atom-resolution are intrinsically time-consuming, many interesting phenomena are excluded from real-time NMR analysis. Recently, spatially encoded ultrafast 2D NMR techniques have been proposed that allow one to acquire a 2D NMR experiment within a single transient. In addition, when combined with the SOFAST technique, such ultrafast experiments can be repeated at high rates. One of the problems detected for such ultrafast protein NMR experiments is related to the heteronuclear decoupling during detection with interferences between the pulses and the oscillatory magnetic field gradients arising in this scheme. Here we present a method for improved ultrafast data acquisition yielding higher signal to noise and sharper lines in single-scan 2D NMR spectra. In combination with a fast-mixing device, the recording of ¹H–¹⁵N correlation spectra with repetition rates of up to a few Hertz becomes feasible, enabling real-time studies of protein kinetics occurring on time scales down to a few seconds.     

Pressure-Temperature Analysis of the Stability of the CTL9 Domain Reveals Hidden Intermediates

The observation of two-state unfolding for many small single-domain proteins by denaturants has led to speculation that protein sequences may have evolved to limit the population of partially folded states that could be detrimental to fitness. How such strong cooperativity arises from a multitude of individual interactions is not well understood. Here, we investigate the stability and folding cooperativity of the C-terminal domain of the ribosomal protein L9 in the pressure-temperature plane using site-specific NMR. In contrast to apparent cooperative unfolding detected with denaturant-induced and thermal-induced unfolding experiments and stopped-flow refolding studies at ambient pressure, NMR-detected pressure unfolding revealed significant deviation from two-state behavior, with a core region that was selectively destabilized by increasing temperature. Comparison of pressure-dependent NMR signals from both the folded and unfolded states revealed the population of at least one invisible excited state at atmospheric pressure. The core destabilizing cavity-creating I98A mutation apparently increased the cooperativity of the loss of folded-state peak intensity while also increasing the population of this invisible excited state present at atmospheric pressure. These observations highlight how local stability is subtly modulated by sequence to tune protein conformational landscapes and illustrate the ability of pressure- and temperature-dependent studies to reveal otherwise hidden states.     

Tunable fluorescence emission in pyrene-(2,2'-bipyridine) dyads containing phenylene-ethynylene bridges.

A synthetic route is described for the preparation of a series of pyrene-containing pi-conjugated 2,2'-bipyridine (bpy) ligands. These compounds have been investigated by steady-state and time-resolved fluorescence spectroscopy. They display intense visible absorption and fluorescence emission properties that can be very efficiently modulated by the complexation of zinc(ii) metal ions to the bpy coordinating unit. The solvatochromism of the emission band of the zinc(ii) complexes, the fluorescence quantum yields, and lifetimes in THF have been determined. Zinc(ii)-induced formation of a charge transfer singlet excited states induces an increase in dipole moment of more than 20 D. Semiempirical theoretical calculations were performed and allowed to assess the electronic nature of ground and excited states of the free ligands and and that of the corresponding zinc(ii) complexes.     

Probing ground and excited states of phospholamban in model and native lipid membranes by magic angle spinning NMR spectroscopy

In this paper, we analyzed the ground and excited states of phospholamban (PLN), a membrane protein that regulates sarcoplasmic reticulum calcium ATPase (SERCA), in different membrane mimetic environments. Previously, we proposed that the conformational equilibria of PLN are central to SERCA regulation. Here, we show that these equilibria detected in micelles and bicelles are also present in native sarcoplasmic reticulum lipid membranes as probed by MAS solid-state NMR. Importantly, we found that the kinetics of conformational exchange and the extent of ground and excited states in detergent micelles and lipid bilayers are different, revealing a possible role of the membrane composition on the allosteric regulation of SERCA. Since the extent of excited states is directly correlated to SERCA inhibition, these findings open up the exciting possibility that calcium transport in the heart can be controlled by the lipid bilayer composition. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Thermodynamic and kinetic exploration of the energy landscape of Borrelia burgdorferi OspA by native-state hydrogen exchange

We report a native-state hydrogen-exchange (HX) method to simultaneously obtain both thermodynamic and kinetic information on the formation of multiple excited states in a folding energy landscape. Our method exploits the inherent dispersion and pH dependence of the intrinsic HX rates to cover both the EX2 (thermodynamic) and EX1 (kinetic) regimes. At each concentration of denaturant, HX measurements are performed over a range of pH values. Using this strategy, we dissected Borrelia burgdorferi OspA, a predominantly beta-sheet protein containing a unique single-layer beta-sheet, into five cooperative units and postulated excited states predominantly responsible for HX. More importantly, we determined the interconversion rates between these excited states and the native state. The use of both thermodynamic and kinetic information from native-state HX enabled us to construct a folding landscape of this 28kDa protein, including local minima and maxima, and to discriminate on-pathway and off-pathway intermediates. This method, which we term EX2/EX1 HX, should be a powerful tool for characterizing the complex folding mechanisms exhibited by the majority of proteins.     

The low energy emitting states of the Lhca4 subunit of higher plant photosystem I

The selectively red excited emission spectrum, at room temperature, of the in vitro reconstituted Lhca4, has a pronounced non-equilibrium distribution, leading to enhanced emission from the directly excited low-energy pigments. Two different emitting forms (or states), with maximal emission at 713 and 735nm (F713 and F735) and unusual spectral properties, have been identified. Both high-energy states are populated when selective excitation is into the F735 state and the fluorescence anisotropy spectrum attains the value of 0.3 in the wavelength region where both emission states are present. This indicates that the two states are on the same Lhca4 complex and have transition dipoles with similar orientation.     

Thermodynamics of protein denatured states

Recent work on the thermodynamics of protein denatured states is providing insight into the stability of residual structure and the conformational constraints that affect the disordered states of proteins. Current data from native state hydrogen exchange and the pH dependence of protein stability indicate that residual structure can modulate the stability of the denatured state by up to 4 kcal mol(-1). NMR structural data have emphasized the role of hydrophobic clusters in stabilizing denatured state residual structures, however recent results indicate that electrostatic interactions, both favorable and unfavorable, are also important modulators of the stability of the denatured state. Thermodynamics methods that take advantage of histidine-heme ligation chemistry have also been developed to probe the conformational constraints that act on denatured states. These methods have provided insights into the role of excluded volume, chain stiffness, and loop persistence in modulating the conformational preferences of highly disordered proteins. New insights into protein folding and novel methods to manipulate protein stability are emerging from this work.     

The photophysical behavior of mixed transition metal-uranyl complexes with polyketonate ligands. The vibronically isolated uranyl chromophore

The uranyl excited-state lifetimes and luminescence spectra have been examined for a series of bis- triketonate and bis-tetraketonate uranyl— transition metal complexes at low temperatures. The energies of the vibronic components of the uranyl luminescence were found to be dependent on the complexing ligand, but they did not depend significantly on the neighboring transition metal (Cu, Co, Fe, Ni, Zn, Pd). The band shape was sometimes markedly dependent on the metal. Emission quantum yields varied over a 100-fold range. Emission lifetimes varied by less than a factor of three, despite the fact that most of the transition metals are potential quenchers, and despite the existence of a low energy ligand-to-metal charge-transfer excited state in the tetraketonate complexes. The vibronic isolation of the uranyl excited state from other molecular excited states in these complexes is attributed to a large nuclear reorganizational barrier for entry into or escape from the potential energy surface of the electronically excited uranyl moiety. Population of the uranyl excited state results in an increase in the UO bond length, and the UO nuclear motions are not activated by other low energy electronic excited states of the polyketonate complexes.     

Electronic Excited States of the CP29 Antenna Complex of Green Plants: A Model Based on Exciton Calculations

We have suggested a model for the electronic excited states of the minorplant antenna, CP29, by incorporating a considerable part of the currentinformation offered by structure determination, site-directed mutagenesis,and spectroscopy in the modeling.We have assumed that the electronic excited states of the complex havebeen decided by the chlorophyll-chlorophyll (Chl) and Chl-proteininteractions and have modeled the Coulombic interaction between a pairof Chls in the point-dipole approximation and the Chl-protein interactionsare treated as empirical fit parameters.We have suggested the Q_y dipole moment orientations and the siteenergies for all the chlorophylls in the complex through a simultaneoussimulation of the absorption and linear dichroism spectra.The assignments proposed have been discussed to yield a satisfactoryreproduction of all prominent features of the absorption, linear and circulardichroism spectra as well as the key spectral and temporal characteristics ofthe energy transfer processes among the chlorophylls.The orientations and the spectral assignments obtained by relatively simpleexciton calculations have been necessary to provide a good point ofdeparture for more detailed treatments of structure-function relationship inCP29. Moreover, it has been discussed that the CP29 model suggested canguide the studies for a better understanding of the structure-functionrelationship in the major plant antenna, LHCII.     

Side-chain interactions in the folding pathway of a Fyn SH3 domain mutant studied by relaxation dispersion NMR spectroscopy

A major challenge to the study of protein folding is the fact that intermediate states along the reaction pathway are generally unstable and thus difficult to observe. Recently developed NMR relaxation dispersion experiments present an avenue to accessing such states, providing kinetic, thermodynamic, and structural information for intermediates with small (greater than or equal to approximately 1%) populations at equilibrium. We have employed these techniques to study the three-state folding reaction of the G48M Fyn SH3 domain. Using (13)C-, (1)H-, and (15)N-based methods, we have characterized backbone and side-chain interactions in the folded, unfolded, intermediate, and transition states, thereby mapping the energy landscape of the protein. We find that the intermediate, populated to approximately 1%, contains nativelike structure in a central beta-sheet, and is disordered at the amino and carboxy termini. The intermediate is stabilized by side-chain van der Waals contacts, yet (13)C chemical shifts indicate that methyl-containing residues remain disordered. This state has a partially structured backbone and a collapsed yet mobile hydrophobic core and thus closely resembles a molten globule. Nonpolar side-chain contacts are formed in the unfolded-intermediate transition state; these interactions are disrupted in the intermediate-folded transition state, possibly allowing side chains to rearrange as they adopt the native packing configuration. This work illustrates the power of novel relaxation dispersion experiments in characterizing excited states that are "invisible" in even the most sensitive of NMR experiments.     

satFRET: estimation of Förster resonance energy transfer by acceptor saturation

We demonstrate theoretically and experimentally the quantification of Förster resonance energy transfer (FRET) by direct and systematic saturation of the excited state of acceptor molecules. This version of acceptor depletion methods for FRET estimation, denoted as “satFRET” is reversible and suitable for time-resolved measurements. The technique was investigated theoretically using the steady-state solution of the differential equation system of donor and acceptor molecular states. The influence of acceptor photobleaching during measurement was included in the model. Experimental verification was achieved with the FRET-pair Alexa 546- Alexa 633 loaded on particles in different stoichiometries and measured in a confocal microscope. Estimates of energy transfer efficiency by excited state saturation were compared to those obtained by measurements of sensitised emission and acceptor photobleaching. The results lead to a protocol that allows time-resolved FRET measurements of fixed and living cells on a conventional confocal microscope. This procedure was applied to fixed Chinese hamster ovary cells containing a cyan fluorescent protein and yellow fluorescent protein pair. The time resolution of the technique was demonstrated in a live T cell activation assay comparing the FRET efficiencies measured using a genetically encoded green and red fluorescent protein biosensor for GTP/GDP turnover to those measured by acceptor photobleaching of fixed cells.