Anjali Pandit

Elucidation of the self-assembly pathway of lanreotide octapeptide into beta-sheet nanotubes: role of two stable intermediates.

Nanofabrication by molecular self-assembly involves the design of molecules and self-assembly strategies so that shape and chemical complementarities drive the units to organize spontaneously into the desired structures. The power of self-assembly makes it the ubiquitous strategy of living organized matter and provides a powerful tool to chemists. However, a challenging issue in the self-assembly of complex supramolecular structures is to understand how kinetically efficient pathways emerge from the multitude of possible transition states and routes. Unfortunately, very few systems provide an intelligible structure and formation mechanism on which new models can be developed. Here, we elucidate the molecular and supramolecular self-assembly mechanism of synthetic octapeptide into nanotubes in equilibrium conditions. Their complex hierarchical self-assembly has recently been described at the mesoscopic level, and we show now that this system uniquely exhibits three assembly stages and three intermediates: (i) a peptide dimer is evidenced by both analytical centrifugation and NMR translational diffusion experiments; (ii) an open ribbon and (iii) an unstable helical ribbon are both visualized by transmission electron microscopy and characterized by small angle X-ray scattering. Interestingly, the structural features of two stable intermediates are related to the final nanotube organization as they set, respectively, the nanotube wall thickness and the final wall curvature radius. We propose that a specific self-assembly pathway is selected by the existence of such preorganized and stable intermediates so that a unique final molecular organization is kinetically favored. Our findings suggests that the rational design of oligopeptides can encode both molecular- and macro-scale morphological characteristics of their higher-order assemblies, thus opening the way to ultrahigh resolution peptide scaffold engineering.

The nature of coherences in the B820 bacteriochlorophyll dimer revealed by two-dimensional electronic spectroscopy

Light-harvesting in photosynthesis is determined by the excitonic interactions in disordered antennae and the coupling of collective electronic excitations to fast nuclear motions, producing efficient energy transfer with a complicated interplay between exciton and vibrational coherences. Two-dimensional electronic spectroscopy (2DES) is a powerful tool to study the presence of these coherences in photosynthetic complexes. However, the unambiguous assignment of the nature of the observed coherences is still under debate. In this paper we apply 2DES to an excitonically coupled bacteriochlorophyll dimer, the B820 subunit of the light harvesting complex 1 (LH1-RC) of R. rubrum G9. Fourier analysis of the measured kinetics and modeling of the spectral responses in a complete basis of electronic and vibrational states allow us to distinguish between pure vibrational, mixed exciton-vibrational (vibronic), and predominantly exciton coherences. The mixed coherences have been found in a wide range of oscillation frequencies, whereas exciton coherences give the biggest contributions for the frequencies in the 400-550 cm(-1) range, corresponding to the exciton splitting energy of the B820 dimer. Significant exciton coherences are also present at higher frequencies, i.e., up to 800 cm(-1), which are determined by realizations of the disorder with a large energy gap between the two pigments (which increases the apparent value of the exciton splitting). Although the B820 dimer is a model system, the approach presented here represents a basis for further analyses of more complicated systems, providing a tool for studying the interplay between electronic and vibrational coherences in disordered photosynthetic antennae and reaction centres.

An NMR comparison of the light-harvesting complex II (LHCII) in active and photoprotective states reveals subtle changes in the chlorophyll a ground-state electronic structures

To protect the photosynthetic apparatus against photo-damage in high sunlight, the photosynthetic antenna of oxygenic organisms can switch from a light-harvesting to a photoprotective mode through the process of non-photochemical quenching (NPQ). There is growing evidence that light-harvesting proteins of photosystem II participate in photoprotection by a built-in capacity to switch their conformation between light-harvesting and energy-dissipating states. Here we applied high-resolution Magic-Angle Spinning Nuclear Magnetic Resonance on uniformly (13)C-enriched major light-harvesting complex II (LHCII) of Chlamydomonas reinhardtii in active or quenched states. Our results reveal that the switch into a dissipative state is accompanied by subtle changes in the chlorophyll (Chl) a ground-state electronic structures that affect their NMR responses, particularly for the macrocycle (13)C4, (13)C5 and (13)C6 carbon atoms. Inspection of the LHCII X-ray structures shows that of the Chl molecules in the terminal emitter domain, where excited-state energy accumulates prior to further transfer or dissipation, the C4, 5 and 6 atoms are in closest proximity to lutein; supporting quenching mechanisms that involve altered Chl-lutein interactions in the dissipative state. In addition the observed changes could represent altered interactions between Chla and neoxanthin, which alters its configuration under NPQ conditions. The Chls appear to have increased dynamics in unquenched, detergent-solubilized LHCII. Our work demonstrates that solid-state Nuclear Magnetic Resonance is applicable to investigate high-resolution structural details of light-harvesting proteins in varied functional conditions, and represents a valuable tool to address their molecular plasticity associated with photoprotection.

Solid-state NMR applied to photosynthetic light-harvesting complexes

This short review describes how solid-state NMR has provided a mechanistic and electronic picture of pigment–protein and pigment–pigment interactions in photosynthetic antenna complexes. NMR results on purple bacterial antenna complexes show how the packing of the protein and the pigments inside the light-harvesting oligomers induces mutual conformational stress. The protein scaffold produces deformation and electrostatic polarization of the BChl macrocycles and leads to a partial electronic charge transfer between the BChls and their coordinating histidines, which can tune the light-harvesting function. In chlorosome antennae assemblies, the NMR template structure reveals how the chromophores can direct their self-assembly into higher macrostructures which, in turn, tune the light-harvesting properties of the individual molecules by controlling their disorder, structural deformation, and electronic polarization without the need for a protein scaffold. These results pave the way for addressing the next challenge, which is to resolve the functional conformational dynamics of the lhc antennae of oxygenic species that allows them to switch between light-emitting and light-energy dissipating states.

Structure Determination of a Bio-Inspired Self-Assembled Light-Harvesting Antenna by Solid-State NMR and Molecular Modeling

The molecular stacking of an artificial light-harvesting antenna self-assembled from 3(1)-aminofunctionalized zinc-chlorins was determined by solid-state NMR in combination with quantum-chemical and molecular-mechanics modeling. A library of trial molecular stacking arrangements was generated based on available structural data for natural and semisynthetic homologues of the Zn-chlorins. NMR assignments obtained for the monomer in solution were validated for self-assembled aggregates and refined with (1)H-(13)C heteronuclear correlation spectroscopy data collected from samples with (13)C at natural abundance. Solid-state ring-current shifts for the (1)H provided spatial constraints to determine the molecular overlap. This procedure allows for a discrimination between different self-assembled structures and a classification of the stacking mode in terms of electric dipole alignment and π-π interactions, parameters that determine the functional properties of light-harvesting assemblies and conducting nanowires. The combination with quantum-mechanical modeling then allowed building a low-resolution packing model in silico from molecular stacks. The method allows for moderate disorder and residual polymorphism at the stack or molecular level and is generally applicable to determine molecular packing structures of aromatic molecules with structural asymmetry, such as is commonly provided by functionalized side chains that serve to tune the self-assembly process.

Proton-Coupled Electron Transfer Constitutes the Photoactivation Mechanism of the Plant Photoreceptor UVR8.

UVR8 is a novel UV-B photoreceptor that regulates a range of plant responses and is already used as a versatile optogenetic tool. Instead of an exogenous chromophore, UVR8 uniquely employs tryptophan side chains to accomplish UV-B photoreception. UV-B absorption by homodimeric UVR8 induces monomerization and hence signaling, but the underlying photodynamic mechanisms are not known. Here, by using a combination of time-resolved fluorescence and absorption spectroscopy from femto- to microseconds, we provide the first experimental evidence for the UVR8 molecular signaling mechanism. The results indicate that tryptophan residues at the dimer interface engage in photoinduced proton coupled electron transfer reactions that induce monomerization.

First solid-state NMR analysis of uniformly 13C-enriched major light-harvesting complexes from Chlamydomonas reinhardtii and identification of protein and cofactor spin clusters

The light-harvesting complex II (LHCII) is the main component of the antenna system of plants and green algae and plays a major role in the capture of sun light for photosynthesis. The LHCII complexes have also been proposed to play a key role in the optimization of photosynthetic efficiency through the process of state 1-state 2 transitions and are involved in down-regulation of photosynthesis under excess light by energy dissipation through non-photochemical quenching (NPQ). We present here the first solid-state magic-angle spinning (MAS) NMR data of the major light-harvesting complex (LHCII) of Chlamydomonas reinhardtii, a eukaryotic green alga. We are able to identify nuclear spin clusters of the protein and of its associated chlorophyll pigments in ¹³C-¹³C dipolar homonuclear correlation spectra on a uniformly ¹³C-labeled sample. In particular, we were able to resolve several chlorophyll 13¹ carbon resonances that are sensitive to hydrogen bonding to the 13¹-keto carbonyl group. The data show that ¹³C NMR signals of the pigments and protein sites are well resolved, thus paving the way to study possible structural reorganization processes involved in light-harvesting regulation through MAS solid-state NMR.

Nuclear Magnetic Resonance Secondary Shifts of a Light-Harvesting 2 Complex Reveal Local Backbone Perturbations Induced by Its Higher-Order Interactions

Protein nuclear magnetic resonance (NMR) secondary chemical shifts are widely used to predict the secondary structure, and in solid-state NMR, they are often the only unambiguous structural parameters available. However, the employed prediction methods are empirical in nature, relying on the assumption that secondary shifts are only affected by shielding effects of neighboring atoms. We analyzed the secondary shifts of a photosynthetic membrane protein with a high density of chromophores and very tight packing, the light-harvesting 2 (LH2) complex of Rhodopseudomonas acidophila. A relation was found between secondary shift anomalies and protein-protein or pigment-protein tertiary and quaternary contacts. For several residues, including the bacteriochlorophyll-coordinating histidines (alphaH31 and betaH30) and the phenylalanine alphaF41 that has strongly twisted C(b)-C(a)-C and C(a)-C-N conformations in the LH2 crystal structure, the perturbing effects on the backbone chemical shifts were tested by density functional theory (DFT) calculations. We propose that higher-order interactions in the tightly packed complex can induce localized perturbations of the backbone conformation and electronic structure, related to functional pigment-protein or protein-protein interactions.

Selective chemical shift assignment of bacteriochlorophyll a in uniformly [13C-15N]-labeled light-harvesting 1 complexes by solid-state NMR in ultrahigh magnetic field.

Magic-angle spinning (MAS) (13)C-(13)C correlation NMR spectroscopy was used to resolve the electronic ground state characteristics of the bacteriochlorophyll a (BChl a) cofactors in light-harvesting 1 (LH1) complexes of Rhodopseudomonas acidophila (strain 10050). The BChl a (13)C isotropic chemical shifts of the LH1 complexes are compared to the (13)C chemical shifts for BChl a dissolved in acetone-d(6) and to (13)C NMR data that has been obtained for the B800 and B850 BChl molecules in Rps. acidophila peripheral light-harvesting complexes (LH2). Since both complexes contain BChl a cofactors, we can address the chemical shift variability for specific carbon responses between the two types of antennae. The global shift pattern of the LH1 BChls resembles the shift patterns of the LH2 alpha- and beta-B850 BChls, while some carbon responses, in particular the C3 and C3(1), show significant deviations. A comparison with density functional theory (DFT) shift calculations provides insight into the BChl concomitant structural and electronic interactions in the ground state. The differences in the LH1 BChl observed chemical shifts relative to the (13)C responses of BChl a in solution cannot be explained by local side chain interactions, such as hydrogen bonding or nonplanarity of the C3 acetyl, but appear to be dominated by protein-induced macrocycle distortion. Such shaping of the macrocycle will contribute significantly to the red shift of the BChl Q(y) absorbance band in purple bacterial light-harvesting complexes.

Ultrafast proton shuttling in Psammocora cyan fluorescent protein

Cyan, green, yellow, and red fluorescent proteins (FPs) homologous to green fluorescent protein (GFP) are used extensively as model systems to study fundamental processes in photobiology, such as the capture of light energy by protein-embedded chromophores, color tuning by the protein matrix, energy conversion by Förster resonance energy transfer (FRET), and excited-state proton transfer (ESPT) reactions. Recently, a novel cyan fluorescent protein (CFP) termed psamFP488 was isolated from the genus Psammocora of reef building corals. Within the cyan color class, psamFP488 is unusual because it exhibits a significantly extended Stokes shift. Here, we applied ultrafast transient absorption and pump-dump-probe spectroscopy to investigate the mechanistic basis of psamFP488 fluorescence, complemented with fluorescence quantum yield and dynamic light scattering measurements. Transient absorption spectroscopy indicated that, upon excitation at 410 nm, the stimulated cyan emission rises in 170 fs. With pump-dump-probe spectroscopy, we observe a very short-lived (110 fs) ground-state intermediate that we assign to the deprotonated, anionic chromophore. In addition, a minor fraction (14%) decays with 3.5 ps to the ground state. Structural analysis of homologous proteins indicates that Glu-167 is likely positioned in sufficiently close vicinity to the chromophore to act as a proton acceptor. Our findings support a model where unusually fast ESPT from the neutral chromophore to Glu-167 with a time constant of 170 fs and resulting emission from the anionic chromophore forms the basis of the large psamFP488 Stokes shift. When dumped to the ground state, the proton on neutral Glu is very rapidly shuttled back to the anionic chromophore in 110 fs. Proton shuttling in excited and ground states is a factor of 20-4000 faster than in GFP, which probably results from a favorable hydrogen-bonding geometry between the chromophore phenolic oxygen and the glutamate acceptor, possibly involving a short hydrogen bond. At any time in the reaction, the proton is localized on either the chromophore or Glu-167, which implies that most likely no low-barrier hydrogen bond exists between these molecular groups. This work supports the notion that proton transfer in biological systems, be it in an electronic excited or ground state, can be an intrinsically fast process that occurs on a 100 fs time scale. PsamFP488 represents an attractive model system that poses an ultrafast proton transfer regime in discrete steps. It constitutes a valuable model system in addition to wild type GFP, where proton transfer is relatively slow, and the S65T/H148D GFP mutant, where the effects of low-barrier hydrogen bonds dominate.