首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
Photoactive yellow protein (PYP) is a prototype of the PAS domain superfamily of signaling proteins. The signaling process is coupled to a three-state photocycle. After the photoinduced trans-cis isomerization of the chromophore, 4-hydroxycinnamic acid (pCA), an early intermediate (pR) is formed, which proceeds to a second intermediate state (pB) on a sub-millisecond time scale. The signaling process is thought to be connected to the conformational changes upon the formation of pB and its recovery to the ground state (pG), but the exact signaling mechanism is not known. Experimental studies of PYP by solution NMR and X-ray crystallography suggest a very flexible protein backbone in the ground as well as in the signaling state. The relaxation from the pR to the pB state is accompanied by the protonation of the chromophore's phenoxyl group. This was found to be of crucial importance for the relaxation process. With the goal of gaining a better understanding of these experimental observations on an atomistic level, we performed five MD simulations on the three different states of PYP: a 1 ns simulation of PYP in its ground state [pG(MD)], a 1 ns simulation of the pR state [pR(MD)], a 2 ns simulation of the pR state with the chromophore protonated (pRprot), a 2 ns simulation of the pR state with Glu46 exchanged by Gln (pRGln) and a 2 ns simulation of PYP in its signaling state [pB(MD)]. Comparison of the pG simulation results with X-ray and NMR data, and with the results obtained for the pB simulation, confirmed the experimental observations of a rather flexible protein backbone and conformational changes during the recovery of the pG from the pB state. The conformational changes in the region around the chromophore pocket in the pR state were found to be crucially dependent on the strength of the Glu46-pCA hydrogen bond, which restricts the mobility of the chromophore in its unprotonated form considerably. Both the mutation of Glu46 with Gln and the protonation of the chromophore weaken this hydrogen bond, leading to an increased mobility of pCA and large structural changes in its surroundings. These changes, however, differ considerably during the pRGln and pRprot simulations, providing an atomistic explanation for the enhancement of the rate constant in the Gln46 mutant. Electronic supplementary material to this article is available at and is accessible for athorized users. Electronic Publication  相似文献   

2.
Themodynamic and transport properties of intermediate states of the photocyclic reaction of photoactive yellow protein (PYP) were studied by a combination of the pulsed laser-induced transient grating (TG), transient lens (TrL), and photoacoustic (PA) spectroscopies from tens of nanoseconds to hundreds of milliseconds. The diffusion coefficients (D) of PYP in the ground state (pG) and of the second intermediate state (pB) were determined by the TG analysis, and it was found that D of pG is about 1.2 times larger than D of pB. At the same time, D at various denatured conditions were measured using guanidine hydrochloride as the denaturant. D of completely unfolded protein is about 0.4 times that of the native form. The enthalpy of pB is estimated to be 60 kJ/mol by the TrL method with an assumption that the volume change of pB is not sensitive to the temperature. Since the enthalpy of the first intermediate state (pR) is as high as 160 kJ/mol, it implies that most of the photon energy is stored as the strain of the protein in pR, and this may be the driving force for the successive reaction to pB. From the temperature dependence of the volume change, the difference in the thermal expansion coefficients between pG and pR was calculated. All of the characteristic features of PYP, the negative volume change, the larger thermal expansion coefficient, and the slower diffusion process, indicate that the intermediate pR and pB are reasonably interpreted in terms of the unfolded (loosened) protein structure.  相似文献   

3.
Photoactive Yellow Protein (PYP), a phototaxis photoreceptor from Ectothiorhodospira halophila, is a small water-soluble protein that iscrystallisable and excellently photo-stable. It can be activated with light(max= 446 nm), to enter a series of transientintermediates that jointly form the photocycle of this photosensor protein.The most stable of these transient states is the signalling state forphototaxis, pB.The spatial structure of the ground state of PYP, pG and the spectralproperties of the photocycle intermediates have been very well resolved.Owing to its excellent chemical- and photochemical stability, also the spatialstructure of its photocycle intermediates has been characterised with X-raydiffraction and multinuclear NMR spectroscopy. Surprisingly, the resultsobtained showed that their structure is dependent on the molecular contextin which they are formed. Therefore, a large range of diffraction-,scattering- and spectroscopic techniques is now being employed to resolvein detail the dynamical changes of the structure of PYP while it progressesthrough its photocycle. This approach has led to considerable progress,although some techniques still result in mutually inconsistent conclusionsregarding aspects of the structure of particular intermediates.Recently, significant progress has also been made with simulations withmolecular dynamics analyses of the initial events that occur in PYP uponphoto activation. The great challenge in this field is to eventually obtainagreement between predicted dynamical alterations in PYP structure, asobtained with the MD approach and the actually measured dynamicalchanges in its structure as evolving during photocycle progression.  相似文献   

4.
Fourier transform infrared (FTIR) spectroscopy was applied to the blue-light photoreceptor photoactive yellow protein (PYP) to investigate water structural changes possibly involved in the photocycle of PYP. Photointermediates were stabilized at low temperature, and difference IR spectra were obtained between intermediate states and the original state of PYP (pG). Water structural changes were never observed in the >3570 cm(-)(1) region for the intermediates stabilized at 77-250 K, such as the red-shifted pR and blue-shifted pB intermediates. In contrast, a negative band was observed at 3658 cm(-)(1) in the pB minus pG spectrum at 295 K, which shifts to 3648 cm(-)(1) upon hydration with H(2)(18)O. The high frequency of the O-H stretch of water indicates that the water O-H group does not form hydrogen bonds in pG, and newly forms these upon pB formation at 295 K, but not at 250 K. Among 92 water molecules in the crystal structure of PYP, only 1 water molecule, water-200, is present in a hydrophobic core inside the protein. The amide N-H of Gly-7 and the imidazole nitrogen atom of His-108 are its possible hydrogen-bonding partners, indicating that one O-H group of water-200 is free to form an additional hydrogen bond. The water band at 3658 cm(-)(1) was indeed diminished in the H108F protein, which strongly suggests that the water band originates from water-200. Structural changes of amide bands in pB were much greater in the wild-type protein at 295 K than at 250 K or in the H108F protein at 295 K. The position of water-200 is >15 A remote from the chromophore. Virtually no structural changes were reported for regions larger than a few angstroms away from the chromophore, in the time-resolved X-ray crystallography experiments on pB. On the basis of the present results, as well as other spectroscopic observations, we conclude that water-200 (buried in a hydrophobic core in pG) is exposed to the aqueous phase upon formation of pB in solution. In neither crystalline PYP nor at low temperature is this structural transition observed, presumably because of the restrictions on global structural changes in the protein under these conditions.  相似文献   

5.
The nature of the pB intermediate of photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been probed by NMR. pH-dependent changes in the NMR spectrum of the dark state of PYP are shown to closely mimic exchange broadening effects observed previously in the NMR spectrum of the pB intermediate in solution. Amide H-D exchange data show that while pB retains a solid protected core, two regions become significantly less protected than the dark state. The amide exchange data help to rationalize why the conformational exchange process affects the N-terminal 28-residue segment of the protein, which is not close to the site of chromophore rearrangement. At very low pH (pH 1.7), the dark state NMR spectrum displays approximately 30 very sharp signals, which are characteristic of a portion of the molecule becoming unfolded. Similarities between the dark state spectra at pH approximately 3.2 and the spectra of pB suggest a model for pB in solution where the protein exists in an equilibrium between a well-ordered state and a state in which a region is unfolded. Such a two-state model accounts for the exchange phenomena observed in the NMR spectra of pB, and the hydrophobic exposure and lability inferred from thermodynamic data. It is likely that in the crystalline environment the ordered form of pB is strongly favored.  相似文献   

6.
As a bacterial blue light sensor the photoactive yellow protein (PYP) undergoes conformational changes upon signal transduction. The absorption of a photon triggers a series of events that are initially localized around the protein chromophore, extends to encompass the whole protein within microseconds, and leads to the formation of the transient pB signaling state. We study the formation of this signaling state pB by molecular simulation and predict its solution structure. Conventional straightforward molecular dynamics is not able to address this formation process due to the long (microsecond) timescales involved, which are (partially) caused by the presence of free energy barriers between the metastable states. To overcome these barriers, we employed the parallel tempering (or replica exchange) method, thus enabling us to predict qualitatively the formation of the PYP signaling state pB. In contrast to the receptor state pG of PYP, the characteristics of this predicted pB structure include a wide open chromophore-binding pocket, with the chromophore and Glu(46) fully solvent-exposed. In addition, loss of alpha-helical structure occurs, caused by the opening motion of the chromophore-binding pocket and the disruptive interaction of the negatively charged Glu(46) with the backbone atoms in the hydrophobic core of the N-terminal cap. Recent NMR experiments agree very well with these predictions.  相似文献   

7.
The change in the electrostatic properties on excitation of the cofactor of wild-type photoactive yellow protein (WT-PYP) have been directly determined using Stark-effect spectroscopy. We find that, instantaneously on photon absorption, there is a large change in the permanent dipole moment, /Delta(-->)mu/, (26 Debye) and in the polarizability, (-)Deltaalpha, (1000 A(3)). We expect such a large degree of charge motion to have a significant impact on the photocycle that is associated with the important blue-light negative phototactic response of Halorhodospira halophila. Furthermore, changing E46 to Q in WT-PYP does not significantly alter its electrostatic properties, whereas, altering the chromophore to prevent it from undergoing trans-cis isomerization results in a significant diminution of /Delta(-->)mu/ and (-)Deltaalpha. We propose that the enormous charge motion that occurs on excitation of 4-hydroxycinnamyl thioester, the chromophore in WT-PYP, plays a crucial role in initiating the photocycle by translocation of the negative charge, localized on the phenolate oxygen in the ground state, across the chromophore. We hypothesize that this charge motion would consequently increase the flexibility of the thioester tail thereby decreasing the activation barrier for the rotation of this moiety in the excited state.  相似文献   

8.
The Photoactive Yellow Protein (PYP) from Halorhodospira halophila (formerly Ectothiorhodospira halophila) is increasingly used as a model system. As such, a thorough understanding of the photocycle of PYP is essential. In this study we have combined information from pOH- (or pH-) dependence and (kinetic) deuterium isotope effects to elaborate on existing photocycle models. For several characteristics of PYP we were able to make a distinction between pH- and pOH-dependence, a nontrivial distinction when comparing data from samples dissolved in H2O and D2O. It turns out that most characteristics of PYP are pOH-dependent. We confirmed the existence of a pB′ intermediate in the pR to pB transition of the photocycle. In addition, we were able to show that the pR to pB′ transition is reversible, which explains the previously observed biexponential character of the pR-to-pB photocycle step. Also, the absorption spectrum of pB′ is slightly red-shifted with respect to pB. The recovery of the pG state is accompanied by an inverse kinetic deuterium isotope effect. Our interpretation of this is that before the chromophore can be isomerized, it is deprotonated by a hydroxide ion from solution. From this we propose a new photocycle intermediate, pBdeprot, from which pG is recovered and which is in equilibrium with pB. This is supported in our data through the combination of the observed pOH and pH dependence, together with the kinetic deuterium isotope effect.  相似文献   

9.
The energetics, protein dynamics, and diffusion coefficients of three mutants of photoactive yellow protein, R52Q, P68A, and W119G, were studied by the transient grating and pulsed laser-induced photoacoustic method. We observed a new dynamics with a lifetime of approximately 1 micro s in the transient grating signal, which is silent by the light absorption technique. This fact indicates that, after the structure change around the chromophore is completed (pR(1)), the protein part located far from the chromophore is still moving to finally create another pR (pR(2)) species, which can transform to the next intermediate, pB. Although the kinetics of pR(2)-->pB-->pG are very different depending on the mutants, the enthalpies of the first long-lived (in micro seconds, 100-micro s range) intermediate species (pR(2)) are similar and very high for all mutants. The diffusion coefficients of the parent (pG) and pB species of the mutants are also similar to that of the wild-type photoactive yellow protein. From the temperature dependence of the volume change, the difference in the thermal expansion coefficients taken as indicator of the flexibility of the structure between pG and pR(2) is measured. They are also similar to that of the wild-type photoactive yellow protein. These results suggest that the protein structures of pR(2) and pB in these mutants are globally different from that of pG, and this structural change is not altered so much by the single amino acid residue mutation. This is consistent with the partially unfolded nature of these intermediate species. On the other hand, the volume changes during pR(1)-->pR(2) are sensitive to the mutations, which may suggest that the volume change reflects a rather local character of the structure, such as the chromophore-protein interaction.  相似文献   

10.
11.
The photocycle of the blue-light photoreceptor protein Photoactive Yellow Protein (PYP) was studied at reduced relative humidity (RH). Photocycle kinetics and spectra were measured in thin films of PYP in which the relative humidity was set at values between 29 and 98% RH with saturated solutions of various salts. We show that in this range, approximately 200 water molecules per PYP molecule are released from the film. As humidity decreased, photocycle transition rates changed, until at low humidity (RH < 50%) an authentic photocycle was no longer observed and the absorption spectrum of the dark, equilibrium state of PYP started to shift to 355 nm, that is, to a form resembling that of pB(dark). At moderately reduced humidity (i.e., >50% RH), an authentic photocycle is still observed, although its characteristics differ from those in solution. As humidity decreases, the rate of ground state recovery increases, while the rate of depletion of the first red-shifted intermediate pR dramatically decreases. The latter observation contrasts all so-far known modulations of the rate of the transition of the red-shifted- to the blue-shifted intermediates of PYP, which is consistently accelerated by all other modulations of the mesoscopic context of the protein. Under these same conditions, the long-lived, blue-shifted intermediate was formed not only with slower kinetics than in solution but also to a smaller extent. Global analysis of these data indicates that in this low humidity environment the photocycle can take a different route than in solution, that is, part of pG recovers directly from pR. These experiments on wild-type PYP, in combination with observations on a variant of PYP obtained by site-directed mutagenesis (the E46Q mutant protein), further document the context dependence of the photocycle transitions of PYP and are relevant for the interpretation of results obtained in both spectroscopic and diffraction studies with crystalline PYP.  相似文献   

12.
Photoactive yellow protein, a small, water-soluble blue-light absorbing photoreceptor protein from Ectothiorhodospira(Halorhodospira)[space]halophila has a structure with two hydrophobic cores, of which the main one houses its light-sensitive chromophore (p-coumaric acid), separated by a central [small beta]-sheet. This photoreceptor protein contains a single tryptophan residue (W119) that is situated at the interface between the central beta-sheet and its N-terminal cap. The fluorescence properties of W119 in the dark state pG (lambda(max)= 328 nm; Phi(fl)= 0.01; nearly pH-independent) are typical for a buried tryptophan in a hydrophobic environment with significant quenching by nearby amino acid residues. Signalling state formation leads to pH-dependent fluorescence changes: At pH values <6.5 the fluorescence emission increases, with a minor blue shift of the emission maximum. Above this pH, the emission maximum of the tryptophan shifts considerably to the red, whereas its total intensity decreases. These results further support the contention that signalling state formation in PYP leads to significant changes in the structure of this protein, even at sites that are at a considerable distance from the chromophore. The nature of these changes in pB, however, depend upon the pH imposed upon the protein: At slightly alkaline pH, which presumably is closest to the pH to which this protein is exposed in vivo, these changes lead to an exposure of the part of the central beta-sheet harbouring W119. At slightly acidic pH the polarity of the environment of W119 is hardly affected by the formation of the signalling state but the quenching of its fluorescence emission, possibly by nearby amino acids, is reduced. On the other hand, its accessibility for quenching by small molecules in the solution is enhanced at acidic and alkaline pH in the signalling state (pB) compared to the dark state (pG). This latter observation points towards a more flexible structure of the N-terminal cap, having a looser interaction with the central beta-sheet in pB.  相似文献   

13.
Light-dependent pH changes were measured in unbuffered solutions of wild type photoactive yellow protein (PYP) and its H108F and E46Q variants, using two independent techniques: transient absorption changes of added pH indicator dyes and direct readings with a combination pH electrode. Depending on the absolute pH of the sample, a reversible protonation as well as a deprotonation can be observed upon formation of the transient, blue-shifted photocycle intermediate (pB) of this photoreceptor protein. The latter is observed at very alkaline pH, the former at acidic pH values. At neutral pH, however, the formation of the pB state is not paralleled by significant protonation/deprotonation of PYP, as expected for concomitant protonation of the chromophore and deprotonation of Glu-46 during pB formation. We interpret these results as further evidence that a proton is transferred from Glu-46 to the coumaric acid chromophore of PYP, during pB formation. One cannot exclude the possibility, however, that this transfer proceeds through the bulk aqueous phase. Simultaneously, an amino acid side chain(s) (e.g. His-108) changes from a buried to an exposed position. These results, therefore, further support the idea that PYP significantly unfolds in the pB state and resolve the controversy regarding proton transfer during the PYP photocycle.  相似文献   

14.
Visualizing the three-dimensional structures of a protein during its biological activity is key to understanding its mechanism. In general, protein structure and function are pH-dependent. Changing the pH provides new insights into the mechanisms that are involved in protein activity. Photoactive yellow protein (PYP) is a signaling protein that serves as an ideal model for time-dependent studies on light-activated proteins. Its photocycle is studied extensively under different pH conditions. However, the structures of the intermediates remain unknown until time-resolved crystallography is employed. With the newest beamline developments, a comprehensive time series of Laue data can now be collected from a single protein crystal. This allows us to vary the pH. Here we present the first structure, to our knowledge, of a short-lived protein-inhibitor complex formed in the pB state of the PYP photocycle at pH 4. A water molecule that is transiently stabilized in the chromophore active site prevents the relaxation of the chromophore back to the trans configuration. As a result, the dark-state recovery is slowed down dramatically. At pH 9, PYP stops cycling through the pB state altogether. The electrostatic environment in the chromophore-binding site is the likely reason for this altered kinetics at different pH values.  相似文献   

15.
Stark (electroabsorption) spectra of the M100A mutant of photoactive yellow protein reveal that the neutral, cis cofactor of the pB intermediate undergoes a strikingly large change in the static dipole moment (|Deltamu| = 19 Debye) on photon absorption. The formation of this charge-separated species, in the excited state, precedes the cis --> trans isomerization of the pB cofactor and the regeneration of pG. The large |Deltamu|, reminiscent of that produced on the excitation of pG, we propose, induces twisting of the cis cofactor as a result of translocation of negative charge, from the hydroxyl oxygen, O1, toward the C7-C8 double bond. The biological significance of this photoinduced charge transfer reaction underlies the significantly faster regeneration of pG from pB in vitro, on the absorption of blue light.  相似文献   

16.
We have studied the kinetics of the blue light-induced branching reaction in the photocycle of photoactive yellow protein (PYP) from Ectothiorhodospira halophila, by nanosecond time-resolved UV/Vis spectroscopy. As compared to the parallel dark recovery reaction of the presumed blue-shifted signaling state pB, the light-induced branching reaction showed a 1000-fold higher rate. In addition, a new intermediate was detected in this branching pathway, which, compared to pB, showed a larger extinction coefficient and a blue-shifted absorption maximum. This substantiates the conclusion that isomerization of the chromophore is the rate-controlling step in the thermal photocycle reactions of PYP and implies that absorption of a blue photon leads to cis-->trans isomerization of the 4-hydroxy-cinnamyl chromophore of PYP in its pB state.  相似文献   

17.
Conformational changes in the light illuminated intermediate (pB) of photoactive yellow protein (PYP) were studied from a viewpoint of the diffusion coefficient (D) change of several N-truncated PYPs, which lacked the N-terminal 6, 15, or 23 amino acid residues (T6, T15, and T23, respectively). For intact PYP (i-PYP), D of pB (D(pB)) was approximately 11% lower than that (D(pG)) of the ground state (pG) species. The difference in D (D(pG) - D(pB)) decreased upon cleavage of the N-terminal region in the order of i-PYP>T6>T15>T23. This trend clearly showed that conformational change in the N-terminal group is the main reason for the slower diffusion of pB. This slower diffusion was interpreted in terms of the unfolding of the two alpha-helices in the N-terminal region, increasing the intermolecular interactions due to hydrogen bonding with water molecules. The increase in friction per one residue by the unfolding of the alpha-helix was estimated to be 0.3 x 10(-12) kg/s. The conformational change in the N-terminal group upon photoillumination is discussed.  相似文献   

18.
The long-lived light-induced intermediate (pB) of the E46Q mutant (glutamic acid is replaced by glutamine at position 46) of photoactive yellow protein (PYP) has been investigated by NMR spectroscopy. The ground state of this mutant is very similar to that of wild-type PYP (WT), whereas the pB state, formed upon illumination, appears to be much more structured in E46Q than in WT. The differences are most striking in the N-terminal domain of the protein. In WT, the side-chain carboxylic group of E46 is known to donate its proton to the chromophore upon illumination. The absence of the carboxylic group near the chromophore in the E46Q mutant prohibits the formation of a negative charge at this position upon formation of pB. This prevents the partial unfolding of the mutant, as evidenced from NMR chemical shift comparison and proton/deuterium (H/D) exchange studies.  相似文献   

19.
Proteins involved in signal transduction can usually be present in two states: an inactive and an active (signaling) state. In the case of photoreceptors such as PYP, it has been shown that the signaling state has a large degree of structural and dynamic disorder. Conventional structural NMR approaches present difficulties in describing such partially unfolded states. Owing to the disordered dynamical and transient nature of such states classical NOE-based information, when present, is sparse. Chemical shift changes upon partial unfolding can, however, be easily monitored from HSQC spectra. We show here that such states can be modeled by defining native-like inter-residue contacts for those residues that do not shift significantly upon partial unfolding. The feasibility of this approach is demonstrated using lysozyme as a test case and applied to model the partially unfolded signaling state (pB) of a truncated form of the photoactive yellow protein for which a “classical” NOE-based structure is available for validation. This approach should be generally applicable to systems in which part of the structure remains in a well-defined native-like conformation.  相似文献   

20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号