Protein Nanospace Research Articles

Protein–cofactor binding and ultrafast electron transfer in riboflavin binding protein under the spatial confinement of nanoscopic reverse micelles

In this contribution, we study the effect of confinement on the ultrafast electron transfer (ET) dynamics of riboflavin binding protein (RBP) to the bound cofactor riboflavin (Rf, vitamin B2), an important metabolic process, in anionic sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles (AOT-RMs) of various hydration levels. Notably, in addition to excluded volume effect, various nonspecific interactions like ionic charge of the confining surface can influence the biochemical reactions in the confined environment of the cell. To this end, we have also studied the ET dynamics of RBP-Rf complex under the confinement of a cationic hexadecyltrimethylammonium bromide (CTAB) RMs with similar water pool size to the anionic AOT-RMs towards simulating equal restricted volume effect. It has been found that the spatial confinement of RBP in the AOT-RM of w(0) = 10 leads to the loss of its tertiary structure and hence vitamin binding capacity. Although, RBP regains its binding capacity and tertiary structure in AOT-RMs of w(0) ≥ 20 due to its complete hydration, the ultrafast ET from RBP to Rf merely occurs in such systems. However, to our surprise, the ET process is found to occur in cationic CTAB-RMs of similar volume restriction. It is found that under the spatial confinement of anionic AOT-RM, the isoalloxazine ring of Rf is improperly placed in the protein nanospace so that ET between RBP and Rf is not permitted. This anomaly in the binding behaviour of Rf to RBP in AOT-RMs is believed to be the influence of repulsive potential of the anionic AOT-RM surface to the protein. Our finding thus suggests that under similar size restriction, both the hydration and surface charge of the confining volume could have major implication in the intraprotein ET dynamics in real cellular environments.

Construction of an energy transfer system in the bio-nanocup space by heteromeric assembly of gp27 and gp5 proteins isolated from bacteriophage T4

Protein assemblies, such as viruses and ferritins, have been employed as useful molecular templates for the accumulation of organic and inorganic compounds to construct bio-nanomaterials. While several methods for conjugation of heterofunctional molecules with protein assemblies have been reported, it remains difficult to control their fixation sites in the assemblies. In this article, we demonstrate the three-dimensional arrangement of different types of fluorescent probes using the heteromeric self-assembly of (gp27-gp5)(3) which is the component protein of bacteriophage T4 (gp: gene product). The composites exhibited fluorescence resonance energy transfer from fluorescein to tetramethylrhodamine dyes immobilized in the bio-nanocup space. The alternation of the donor and acceptor positions induced fluorescence self-quenching by the formation of ground-state complexes of the acceptors. These results indicate that the site-specific conjugation method using the bio-nanocup space of the heteromeric protein assembly has potential for the integration of several types of functional molecules in protein nanospaces.

Ultrafast light-induced response of photoactive yellow protein chromophore analogues

The fluorescence decays of several analogues of the photoactive yellow protein (PYP) chromophore in aqueous solution have been measured by femtosecond fluorescence up-conversion and the corresponding time-resolved fluorescence spectra have been reconstructed. The native chromophore of PYP is a thioester derivative of p-coumaric acid in its trans deprotonated form. Fluorescence kinetics are reported for a thioester phenyl analogue and for two analogues where the thioester group has been changed to amide and carboxylate groups. The kinetics are compared to those we previously reported for the analogues bearing ketone and ester groups. The fluorescence decays of the full series are found to lie in the 1-10 ps range depending on the electron-acceptor character of the substituent, in good agreement with the excited-state relaxation kinetics extracted from transient absorption measurements. Steady-state photolysis is also examined and found to depend strongly on the nature of the substituent. While it has been shown that the ultrafast light-induced response of the chromophore in PYP is controlled by the properties of the protein nanospace, the present results demonstrate that, in solution, the relaxation dynamics and pathway of the chromophore is controlled by its electron donor-acceptor structure: structures of stronger electron donor-acceptor character lead to faster decays and less photoisomerisation.

Investigations into the dynamics and mechanisms of ultrafast photoinduced reactions taking place in photoresponsive protein nanospaces (PNS)

Investigations into the dynamics and mechanisms of ultrafast photoinduced reactions taking place in photoresponsive protein nanospaces (PNS)

Ultrafast photoreactions in protein nanospaces as revealed by fs fluorescence dynamics measurements on photoactive yellow protein and related systemsDedicated to Professor Dr Z. R. Grabowski and Professor Dr J. Wirz on the occasions of their 75th and 60th birthdays.

We have investigated primary processes of ultrafast photoreactions of various photoresponsive proteins by means of fs fluorescence dynamics measurements. Based on these studies, the effects of the protein nanospace (PNS), containing the chromophore, on the dynamics and mechanisms of the ultrafast and highly efficient reactions of these proteins have been elucidated. In this article, we discuss mainly the results of our studies on ultrafast photoisomerization of photoactive yellow protein (PYP), its mutants and analogues. The chromophore of the PYP, deprotonated coumaric acid thioester (O−-phenyl-CHCH–CO–S–CH2–), fixed in PNS by hydrogen (H) bonding interactions at the head part, O−-phenyl-, and by covalent bonding at the tail part, –CO–S–, undergoes ultrafast twisting by flipping the thioester bond, owing to the intrachromophore head to tail charge transfer caused by the photoexcitation. In the site-directed mutants where the PNS structure is looser and more disordered, the photoinduced twisting reaction becomes slowed compared with the wild-type PYP and moreover, the twisting becomes much slower in the denatured PYP, showing the supreme importance of more regulated PNS for the fast twisting. We found also coherent vibrations in the fluorescence decay curves which might be coupled with the twisting. Furthermore, we found for a PYP analogue with replaced chromophore ultrafast dynamic Stokes shift of fluorescence rather than the quenching due to twisting, indicating the importance of chromophore-PNS fine adjustment for the ultrafast twisting.

Ultrafast Photoinduced Reaction Dynamics and Coherent Oscillations in Photoresponsive Proteins.

We have studied primary processes of ultrafast photoisomerization reactions of photoactive yellow protein (PYP) and visual rhodopsin (Rh) as well as ultrafast photoinduced electron transfer reactions of flavoproteins (FP) by means of the fs fluorescence up-conversion method. On the basis of these studies, we have examined the effects of the protein nanospace (PNS), where the chromophore is held, on the dynamics and mechanisms of the ultrafast and highly efficient photoinduced reactions of these proteins. In this article, we discuss mainly results of our comparative fluorescence dynamics studies on wild type (w. -t.) PYP, it’s site-directed mutants, analogues with replaced chromophore and FP, including the coherent vibrations in the ultrafast fluorescence decays of w. -t. PYP and mutants in the course of the photoisomerization, ultrafast dynamic Stokes shift of fluorescence of PYP analogues, and ultrafast electron transfer of FP.

Femtosecond Fluorescence Dynamics of Flavoproteins: Comparative Studies on Flavodoxin, Its Site-Directed Mutants, and Riboflavin Binding Protein Regarding Ultrafast Electron Transfer in Protein Nanospaces

We have studied the fluorescence dynamics of “nonfluorescent” flavoproteins including flavodoxin (FD), its mutants W60F, Y98F, and W60F/Y98F, and riboflavin binding protein (RBP) with the femtosecond fluorescence up-conversion method and have observed the fluorescence quenching dynamics of FD and its mutants for the first time. The strong fluorescence quenching in these flavoproteins seems to be caused by ultrafast electron transfer (ET) from aromatic amino acid residues to the excited flavin chromophore in stacked configuration according to previous transient absorption studies. In the present work, we have made comparative studies on the dynamics of fluorescence quenching due to ET to the excited chromophore in RBP and FD. We have observed also fluorescence dynamics of FD mutants where active electron donors Trp·NH and Tyr·OH are partially (either of them) or completely replaced by inactive phenylalanine and directly demonstrated the ET mechanism of the ultrafast fluorescence quenching in PNS of FD.

Ultrafast photoinduced reaction dynamics of photoactive yellow protein (PYP): observation of coherent oscillations in the femtosecond fluorescence decay dynamics

We have observed for the first time coherent oscillations in the fs fluorescence (Fl) dynamics of wild-type (w-t) photoactive yellow protein (PYP) and its site-directed mutants. Two oscillatory modes coupled with the ultrafast Fl decay due to the twisting of the excited chromophore were identified, a high frequency mode (∼140 cm −1) with 700 fs damping time and a low frequency mode (∼50 cm −1) with 500 fs damping time, respectively. The latter becomes overdamped when the structure of the protein nanospace (PNS) surrounding the chromophore becomes more disordered by mutation. Both modes disappear in the Fl dynamics of denatured PYP. Similarities between PYP and rhodopsins (Rh) are discussed.

Dynamics and Mechanisms of Ultrafast Fluorescence Quenching Reactions of Flavin Chromophores in Protein Nanospace

We have studied excited-state dynamics of “nonfluorescent” flavoproteins including riboflavin binding protein (RBP), d-amino acid oxidase benzoate complex (DAOB), and others by means of femtosecond fluorescence up-conversion method and have observed ultrafast fluorescence quenching dynamics for the first time. We have interpreted the fluorescence quenching mechanisms of these flavoproteins as due to the ultrafast electron transfer (ET) to flavin chromophore (F) in the excited electronic state from nearby tryptophan (Trp.NH) or tyrosine (Tyr.OH) residues placed in the protein nanospace (PNS), on the basis of their X-ray structures. Extremely fast fluorescence quenching in RBP (τf ∼ 90−100 fs) could be attributed to the compact stacked arrangement, Trp.NH.....F.....Tyr.OH, supremely favorable for the ultrafast ET reaction dynamics. Comparisons of fluorescence time profiles and spectral characteristics of F in solution with those in PNS have indicated the existence of extremely fast FC (Franck−Condon) → Fl (fluorescence) state conversion in PNS within the time resolution of the apparatus. The ultrafast FC → Fl conversion may be a coherent process coupled with intra-chromophore high-frequency modes leading to formation of vibrationally nonrelaxed or only partially relaxed Fl state, from which barrierless ET seems to occur. Fluorescence dynamics of DAOB have indicated faster initial decay in both blue and red sides of the spectrum contrary to other flavoproteins which showed practically wavelength-independent fluorescence dynamics. This result of DAOB is similar to those of photoactive yellow protein and visual rhodopsin although their reaction mechanism (twisting) is different from DAOB (ET). We have proposed a possible mechanism for this fluorescence dynamics of DAOB on the basis of an extremely compact stacked configuration of F...benzoate-...Tyr.OH which seems to undergo moderate frequency intermolecular vibration coupled with intra-chromophore high-frequency modes of F in the course of ET from Tyr.OH to excited F.

Effects of Modification of Protein Nanospace Structure and Change of Temperature on the Femtosecond to Picosecond Fluorescence Dynamics of Photoactive Yellow Protein

The effect of the protein environment surrounding a chromophore [protein nanospace (PNS)] on the photoinduced ultrafast reaction of the chromophore (deprotonated p-coumaric acid covalently bonded to the protein via a thioester linkage) has been investigated by comparing the femtosecond to picosecond fluorescence dynamics of wild-type (w-t) photoactive yellow protein (PYP) with those of the chromophore in aqueous solution as well as in various PNSs modified by site-directed mutagenesis. The fluorescence decay dynamics of the chromophore are considerably accelerated in PNSs compared with those in solution. The ultrafast nonexponential fluorescence decay dynamics of the w-t PYP in the 100 fs to 10 ps time regime, which are probably due to the formation of a twisted state by the flipping of the thioester linkage of the chromophore, have been slowed in the mutants, which seem to have a looser PNS structure because of the weakening of the chromophore−amino acid residue hydrogen-bonding interaction and the parti...

Environmental Effects on the Femtosecond−Picosecond Fluorescence Dynamics of Photoactive Yellow Protein: Chromophores in Aqueous Solutions and in Protein Nanospaces Modified by Site-Directed Mutagenesis

The effect of the protein environment surrounding chromophores (protein nanospaces) on the photoinduced ultrafast twisting of chromophores in photoactive yellow protein (PYP) was investigated by comparing the femtosecond−picosecond fluorescence dynamics of wild type PYP with those of the chromophore in aqueous solution as well as in protein nanospace modified by site-directed mutagenesis. The rate of the twisted state formation of the chromophore (the primary step of the trans → cis photoisomerization) was demonstrated to be considerably enhanced in protein compared with the aqueous solution. Moreover, results of the measurements of fluorescence dynamics of the chromophore in the protein nanospace modified by site-directed mutagenesis implied that the twisted state formation by flipping the thioester linkage of the chromophore was slowed by modifying the nanospace structure to a slightly looser one. Namely, the more restricted structure of the protein nanospace in the wild-type PYP seems to be best engineered for the twisting by the flipping mechanism.

Ultrafast Fluorescence Quenching Dynamics of Flavin Chromophores in Protein Nanospace

We have studied excited-state dynamics of “nonfluorescent” flavoproteins by means of the femtosecond fluorescence up-conversion method. We have interpreted the ultrafast fluorescence quenching mechanisms of these flavoproteins as due to the ultrafast electron transfer or electron transfer followed by proton-transfer interactions between excited flavin chromophore and nearby tryptophan and tyrosine residues placed in the protein nanospace, on the basis of their X-ray structures. Comparisons of fluorescence time profiles and spectral characteristics of flavin chromophores in solutions with those in protein environments have suggested the existence of extremely fast Franck−Condon → fluorescent state relaxations specific to the protein environments. These results of the ultrafast fluorescence dynamics studies on the nonfluorescent flavoproteins have many features in common with other photobiologically important proteins.