D96N Mutant Research Articles

Propagating Structural Perturbation Inside Bacteriorhodopsin: Crystal Structures of the M State and the D96A and T46V Mutants

The X-ray diffraction structure of the non-illuminated D96A bacteriorhodopsin mutant reveals structural changes as far away as 15 A from residue 96, at the retinal, Trp-182, Ala-215, and waters 501, 402, and 401. The Asp-to-Ala side-chain replacement breaks its hydrogen bond with Thr-46, and the resulting separation of the cytoplasmic ends of helices B and C is communicated to the retinal region through a chain of covalent and hydrogen bonds. The unexpected long-range consequences of the D96A mutation include breaking the hydrogen bond between O of Ala-215 and water 501 and the formation of a new hydrogen bond between water molecules 401 and 402 in the extracellular region. Because in the T46V mutant a new water molecule appears at Asp-96 and its hydrogen-bond to Ile-45 replaces Thr-46 as its link to helix B, the separation of helices B and C is smaller than that in D96A, and there are no atomic displacements elsewhere in the protein. Propagation of conformational changes along the chain between the retinal and Thr-46 had been observed earlier in the crystal structures of the D96N and E204Q mutants but in the trapped M state. Consistent with the perturbation of the retinal region in D96A, little change of the Thr-46 region occurs between the non-illuminated and M states of this mutant. It appears that a local perturbation can propagate along a track in both directions between the retinal and the Asp-96/Thr-46 pair, either from photoisomerization of the retinal in the wild-type protein in one case or from the D96A mutation in the other.

Photoinduced Surface Potential Change of Bacteriorhodopsin Mutant D96N Measured by Scanning Surface Potential Microscopy

We report the direct measurement of photoinduced surface potential differences of wild-type (WT) and mutant D96N bacteriorhodopsin (BR) membranes at pH 7 and 10.5. Atomic force microscopy (AFM) and scanning surface potential microscopy (SSPM) were used to measure the BR membrane with the extracellular side facing up. We present AFM and SSPM images of WT and mutant D96N in which the light-dark transition occurred in the mid-scan of a single BR membrane. Photosteady-state populations of the M state were generated to facilitate measurement in each sample. The photoinduced surface potential of D96N is 63 mV (peak to valley) at pH 10.5 and is 48 mV at pH 7. The photoinduced surface potential of WT is 37 mV at pH 10.5 and approximately 0 at pH 7. Signal magnitudes are proportional to the amount of M produced at each pH. The results indicated that the surface potentials were generated by photoformation of surface charges on the extracellular side of the membrane. Higher surface potential correlated with a longer lifetime of the charges. A mechanistic basis for these signals is proposed, and it is concluded that they represent a steady-state measurement of the B2 photovoltage.

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed 13C NMR

13C NMR spectra of [3- 13C]Ala- and [1- 13C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane α II-helices. Surprisingly, the 13C NMR spectra of [3- 13C]Ala-D85N turned out to be very similar to those of [3- 13C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting 13C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane α II-helices of the M-like state are suppressed already by fluctuation motions in the order of 10 4–10 5 Hz interfered with frequencies of magic angle spinning or proton decoupling. However, 13C NMR signal from the cytoplasmic α-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl 13C NMR signals of the loop and transmembrane α-helices followed by Pro residues in [1- 13C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, 13C NMR spectral feature of reconstituted [1- 13C]Val and [3- 13C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.

Photoisomerization in Proteorhodopsin mutant D97N

The first steps of the photocycle of the D97N mutant of proteorhodopsin (PR) have been investigated by means of ultrafast transient absorption spectroscopy. A comparison with the primary dynamics of native PR and D85N mutant of bacteriorhodopsin is given. Upon photoexcitation of the covalently bound all-trans retinal the excited state decays biexponentially with time constants of 1.4 and 20 ps via a conical intersection, resulting in a 13-cis isomerized retinal. Neither of the two-deactivation channels is significantly preferred. The dynamics is slowed down in comparison with native PR at pH 9 and reaction rates are even lower than for native PR at pH 6, where the primary proton acceptor (Asp97) is protonated. Therefore, the ultrafast isomerization is not only controlled by the charge distribution within the retinal binding pocket. This study shows that in addition to direct electrostatics other effects have to be taken into account to explain the catalytic function of Asp97 in PR on the ultrafast isomerization reaction. This may include sterical interactions and/or bound water molecules within the retinal binding pocket.

Scanning Surface Potential Microscopy of Bacteriorhodopsin Mutant D96N

Scanning Surface Potential Microscopy of Bacteriorhodopsin Mutant D96N

14‐Fluoro‐Bacteriorhodopsin Gelation Films for Dynamic Holography Recording¶

ABSTRACTThe first dynamic holography recording using 14‐fluoro‐(14‐F) bacteriorhodopsin (BR) gelatin films has been achieved. 14‐F BR is an artifical BR pigment made by reconstitution of bacterioopsin (native BR without chromophore) with synthetic 14‐F ratinal. Low‐intensity red light from a cw HE‐Ne laser was used for dynamic holography recording on the 14‐F wild type (WT) BR and 14‐F D96N mutant BR in gelatin films. There is not true comparing the diffraction efficiency for 14‐F D96N BR and 14‐F WT gelatin film, unlike the increase diffraction efficiency for D96N BR gelation film with native chromphore relative to the WT BR gelatin film with native chromophore. Pre‐illumination with blue light of the 14‐F BR gelatin films significantly increases the differaction effciency of both the the 14‐F WT and the 14‐F D96N BR pigments. The sequential application of blue and red laser beams indicates that 14‐F WT and the 14‐F D96N BR pigments. The sequential application of blue and red laser beams indicates taht 14‐F BR gelation films can be useful for optical memory.

Site-directed mutagenesis study of the antibody 2D7 which catalyzes a reaction for insertion of Cu 2+ into mesoporphyrin

Monoclonal antibody 2D7 generated against a transition-state analog N-methyl mesoporphyrin catalyzes a reaction for insertion of a cupric ion into mesoporphyrin. To investigate amino acid residues responsible for the catalytic activity, site-directed mutagenesis of the amino acid residues in the third complementarity determining region of the heavy chain (CDRH3) was performed on the antigen-binding fragment (Fab) of the antibody. Recombinant Fab mutants, in which Arg95 is replaced with Ala (R95A), Asp96 with Asn (D96N) and Met97 with Gly (M97G), were examined in terms of the catalytic efficiency of the reaction (k/K(S)) and the dissociation constant for N-methyl mesoporphyrin binding (K(d)) and these values were compared with those of the wild type. The k/K(S) values of the R95A and D96N mutants were 0.96% and 1.0% of that of the wild type, respectively, whereas the M97G mutant had no detectable catalytic activity. The K(d) values of the R95A and D96N mutants were 165 and 69 times that of the wild type, respectively, while that of the M97G mutant was similar to that of the wild type. The relationship between the k/K(S) and 1/K(d) values in the wild type and the R95A and D96N mutants suggests that Arg95 and Asp96 are responsible for stabilizing the transition-state in the catalytic reaction. The results of the M97G mutant allow us to propose that Met97 plays an important role in the catalytic activity probably due to a subtle and specific conformation of the antibody.

Effect of Dehydration on Photoinduced Transformation in Gelatin Films Made With 14-Fluoro Bacteriorhodopsin Derivatives

Photoinduced transformation in gelatin films made with 14-fluoro bacteriorhodopsin derivatives, both wild-type (WT) and D96N mutant, were studied. Spectral and kinetic peculiarities for these two types of samples were compared over a wide range of relative humidity (9-92%). Analysis of the results considered two existing photoinduced processes that occur in suspensions and films of corresponding pigments. It was demonstrated that there is a range of humidity in which the performance of fluorine WT bacteriorhodopsin gelatin films may offer a technological advantage compared with fluorine D96N bacteriorhodopsin.

2P274 センサリーロドプシンII D75N変異体とトランスデューサータンパク質の相互作用ダイナミクスの研究(光生物 A) 視覚・光受容))

2P274 センサリーロドプシンII D75N変異体とトランスデューサータンパク質の相互作用ダイナミクスの研究(光生物 A) 視覚・光受容))

14-Fluoro-Bacteriorhodopsin Gelatin Films for Dynamic Holography Recording¶

The first dynamic holography recording using 14-fluoro-(14-F) bacteriorhodopsin (BR) gelatin films has been achieved. 14-F BR is an artificial BR pigment made by reconstitution of bacterioopsin (native BR without chromophore) with synthetic 14-F retinal. Low-intensity red light from a cw He-Ne laser was used for dynamic holography recording on the 14-F wild type (WT) BR and 14-F D96N mutant BR in gelatin films. There is not true comparing the diffraction efficiency for 14-F D96N BR and 14-F WT gelatin film, unlike the increased diffraction efficiency for D96N BR gelatin film with native chromophore relative to the WT BR gelatin film with native chromophore. Pre-illumination with blue light of the 14-F BR gelatin films significantly increases the diffraction efficiency of both the 14-F WT and the 14-F D96N BR pigments. The sequential application of blue and red laser beams indicates that 14-F BR gelatin films can be useful for optical memory.

Role of Charged Residues of pharaonis Phoborhodopsin (Sensory Rhodopsin II) in Its Interaction with the Transducer Protein

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, NpSRII) is a receptor for negative phototaxis in Natronomonas (Natronobacterium) pharaonis. In membranes, it forms a 2:2 complex with its transducer protein, pHtrII, which transmits light signals into the cytoplasmic space through protein-protein interactions. We previously found that a specific deprotonated carboxyl of ppR or pHtrII strengthens their binding [Sudo, Y., et al. (2002) Biophys. J. 83, 427-432]. In this study we aim to identify this carboxyl group. Since the D75N mutant has only one photointermediate (ppR(O)(-)(like)) whose existence spans the millisecond time range, the analysis of its decay rate is simple. We prepared various D75N mutants such as D75N/D214N, D75N/K157Q/R162Q/R164Q (D75N/3Gln), D75N/D193N, and D75N/D193E, among which only D75N/D193N did not show pH dependence with regard to the ppR(O)(-)(like) decay rate and K(D) value for binding, implying that the carboxyl group in question is from Asp-193. The pK(a) of this group decreased to below 2 when a complex was formed. Therefore, we conclude that Asp-193(p)()(pR) is connected to the distant transducer-ppR binding surface via hydrogen bonds, thereby modulating its pK(a). In addition, we discuss the importance of Arg-162(p)()(pR) with respect to the binding activity.

Significance of low-frequency local fluctuation motions in the transmembrane B and C alpha-helices of bacteriorhodopsin, to facilitate efficient proton uptake from the cytoplasmic surface, as revealed by site-directed solid-state 13C NMR.

13C NMR spectra of [1-13C]Val- or -Pro-labeled bacteriorhodopsin (bR) and its single or double mutants, including D85N, were recorded at various pH values to reveal conformation and dynamics changes in the transmembrane alpha-helices, in relation to proton release and uptake between bR and the M-like state caused by modified charged states at Asp85 and the Schiff base (SB). It was found that the D85N mutant acquired local fluctuation motion with a frequency of 10(4) Hz in the transmembrane B alpha-helix, concomitant with deprotonation of SB in the M-like state at pH 10, as manifested from a suppressed 13C NMR signal of the [1-13C]-labeled Val49 residue. Nevertheless, local dynamics at Pro50 neighboring with Val49 turned out to be unchanged, irrespective of the charged state of SB as viewed from the 13C NMR of [1-13C]-labeled Pro50. This means that the transmembrane B alpha-helix is able to acquire the fluctuation motion with a frequency of 10(4) Hz beyond the kink at Pro50 in the cytoplasmic side. Concomitantly, fluctuation motion at the C helix with frequency in the order of 10(4) Hz was found to be prominent, due to deprotonation of SB at pH 10, as viewed from the 13C NMR signal of Pro91. Accordingly, we have proposed here a novel mechanism as to proton uptake and transport based on a dynamic aspect that a transient environmental change from a hydrophobic to hydrophilic nature at Asp96 and SB is responsible for the reduced p Ka value which makes proton uptake efficient, as a result of acquisition of the fluctuation motion at the cytoplasmic side of the transmembrane B and C alpha-helices in the M-like state. Further, it is demonstrated that the presence of a van der Waals contact of Val49 with Lys216 at the SB is essential to trigger this sort of dynamic change, as revealed from the 13C NMR data of the D85N/V49A mutant.

Terahertz measurements of the Photoactive Protein Bacteriorhodopsin mutant D96N: M and P states

AbstractWe use terahertz (THz) spectroscopy as a biomaterials characterization tool. Previously we have shown a strong contrast between the THz dielectric response for wild type (WT) and D96N mutant of bacteriorhodopsin. In those studies we observed a large increase in the THz absorbance of WT with excitation to thermally captured photo-intermediates whereas no such increase in absorbance was observed for the mutant D96N. These results suggest that the THz response is sensitive to structural changes and relative flexibility of biomolecules. However the photo-intermediate populations of the WT and D96N samples were not equivalent in those measurements. While the WT samples had relaxed (bR), M and P state intermediates present, the D96N samples had only bR and M states. Here we present terahertz absorbance measurements of D96N as a function of M and P state populations at room temperature. The THz response is constant for intermediate states populations up to 23% M state and up to 30% P state. These results verify that there is a fundamental difference in the conformational dynamics as measured by THz dielectric response for a single residue mutation.

Protein Flexibility and Conformational State: A Comparison of Collective Vibrational Modes of Wild-Type and D96N Bacteriorhodopsin

Far infrared (FIR) spectral measurements of wild-type (WT) and D96N mutant bacteriorhodopsin thin films have been carried out using terahertz time domain spectroscopy as a function of hydration, temperature, and conformational state. The results are compared to calculated spectra generated via normal mode analyses using CHARMM. We find that the FIR absorbance is slowly increasing with frequency and without strong narrow features over the range of 2–60 cm −1 and up to a resolution of 0.17 cm −1. The broad absorption shifts in frequency with decreasing temperature as expected with a strongly anharmonic potential and in agreement with neutron inelastic scattering results. Decreasing hydration shifts the absorption to higher frequencies, possibly resulting from decreased coupling mediated by the interior water molecules. Ground-state FIR absorbances have nearly identical frequency dependence, with the mutant having less optical density than the WT. In the M state, the FIR absorbance of the WT increases whereas there is no change for D96N. These results represent the first measurement of FIR absorbance change as a function of conformational state.

FTIR Spectroscopy of the M Photointermediate in pharaonis Phoborhodopsin

pharaonis phoborhodopsin ( ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the Schiff base of the retinal chromophore is deprotonated upon formation of the M intermediate ( ppR M). The present FTIR spectroscopy of ppR M revealed that the Schiff base proton is transferred to Asp-75, which corresponds to Asp-85 in a light-driven proton-pump bacteriorhodopsin (BR). In addition, the C O stretching vibrations of Asn-105 were assigned for ppR and ppR M. The common hydrogen-bonding alterations in Asn-105 of ppR and Asp-115 of BR were found in the process from photoisomerization (K intermediate) to the primary proton transfer (M intermediate). These results implicate similar protein structural changes between ppR and BR. However, BR M decays to BR N accompanying a proton transfer from Asp-96 to the Schiff base and largely changed protein structure. In the D96N mutant protein of BR that lacks a proton donor to the Schiff base, the N-like protein structure was observed with the deprotonated Schiff base (called M N) at alkaline pH. In ppR, such an N-like (M N-like) structure was not observed at alkaline pH, suggesting that the protein structure of the M state activates its transducer protein.

Stability of Bacteriorhodopsin α-Helices and Loops Analyzed by Single-Molecule Force Spectroscopy

The combination of high-resolution atomic force microscopy imaging and single-molecule force spectroscopy allows the identification, selection, and mechanical investigation of individual proteins. In a recent paper we had used this technique to unfold and extract single bacteriorhodopsins (BRs) from native purple membrane patches. We show that subsets of the unfolding spectra can be classified and grouped to reveal detailed insight into the individualism of the unfolding pathways. We have further developed this technique and analysis to report here on the influence of pH effects and local mutations on the stability of individual structural elements of BR against mechanical unfolding. We found that, although the seven transmembrane α-helices predominantly unfold in pairs, each of the helices may also unfold individually and in some cases even only partially. Additionally, intermittent states in the unfolding process were found, which are associated with the stretching of the extracellular loops connecting the α-helices. This suggests that polypeptide loops potentially act as a barrier to unfolding and contribute significantly to the structural stability of BR. Chemical removal of the Schiff base, the covalent linkage of the photoactive retinal to the helix G, resulted in a predominantly two-step unfolding of this helix. It is concluded that the covalent linkage of the retinal to helix G stabilizes the structure of BR. Trapping mutant D96N in the M state of the proton pumping photocycle did not affect the unfolding barriers of BR.

Redox potentials of the oriented film of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins

The redox potentials of the oriented films of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins (bR), prepared by adsorbing purple membrane (PM) sheets or its mutant on a Pt electrode, have been examined. The redox potentials ( V) of the wild-type bR were −470 mV for the 13- cis configuration of the retinal Shiff base in bR and −757 mV for the all- trans configuration in H 2O, and −433 mV for the 13- cis configuration and −742 mV for the all- trans configuration in D 2O. The solvent isotope effect (Δ V= V(D 2O)− V(H 2O)), which shifts the redox potential to a higher value, originates from the cooperative rearrangements of the extensively hydrogen-bonded water molecules around the protonated CN part in the retinal Schiff base. The redox potential of bR was much higher for the 13- cis configuration than that for the all- trans configuration. The redox potentials for the E194Q mutant in the extracellular region were −507 mV for the 13- cis configuration and −788 mV for the all- trans configuration; and for the E204Q mutant they were −491 mV for the 13- cis configuration and −769 mV for the all- trans configuration. Replacement of the Glu 194 or Glu 204 residues by Gln weakened the electron withdrawing interaction to the protonated CN bond in the retinal Schiff base. The E204 residue is less linked with the hydrogen-bonded network of the proton release pathway compared with E194. The redox potentials of the D96N mutant in the cytoplasmic region were −471 mV for the 13- cis configuration and −760 mV for the all- trans configuration which were virtually the same as those of the wild-type bR, indicating that the D to N point mutation of the 96 residue had no influence on the interaction between the D96 residue and the CN part in the Schiff base under the light-adapted condition. The results suggest that the redox potential of bR is closely correlated to the hydrogen-bonded network spanning from the retinal Schiff base to the extracellular surface of bR in the proton transfer pathway.

Prolonged lifetime of the M intermediate in D96N-mutant bacteriorhodopsin films enhanced by diaza-15-crown-5

In order to get even longer M intermediate lifetime for the purpose of expanding the use of bacteriorhodopsin (BR) materials in optical applications, the combination of genetic engineering and the use of chemical additives was attempted. Different compositions of BR (wild type and D96N mutant)–PVA films with diaza-15-crown-5 (1,4,10-trioxa-7,13-diazacyclopentadecane) additive were prepared. Spectral and kinetic measurements were carried out at room temperature. It was observed that the additive contents that could be used in these films were limited. The highest BR∶diaza-15-crown-5 molecular ratios were 1∶250 and 1∶150 for BRWT and BRD96N films, respectively. Further additions resulted in permanent damage to the purple membrane and a loss of photochromism. The kinetic curves of the M state decay for each film were fit, in a least-squares fashion, by a three-exponential function to obtain sufficiently small residuals. The fitting results indicate that for both types of BR molecules, the decay of the M state was slowed down gradually with increasing additive content. At a BRD96N∶diaza-15-crown-5 molecular ratio of 1∶150, the BR film had the longest lifetime and its photochromism could be observed for about one and a half hours. This indicates that the combination of genetic engineering and chemical additives is more efficient than using either of these two methods. The main reason for this significant prolongation may originate from the strong basic and H+ complexing properties of diaza-15-crown-5 and the change of the membrane surface charge caused by this additive.

Association between a photo-intermediate of a M-lacking mutant D75N of pharaonis phoborhodopsin and its cognate transducer

Pharaonis phoborhodopsin ( ppR or pharaonis sensory rhodopsin II) is a receptor of the negative phototaxis of Natronobacterium pharaonis and forms a complex with its transducer pHtrII in membranes. Flash-photolyis of a D75N mutant did not yield the M-intermediate, but an O-like intermediate is observed in a ms time range. We examined the interaction between the D75N of ppR and t-Htr (truncated pHtrII). These formed a complex in the presence of 0.1% n-dodecyl-β-maltoside, and the association accelerated the decay of the O of D75N from 15 to 56 s −1. From the decay time constants under varying ratios of D75N and t-Htr, n, the molar ratio of D75N/t-Htr in the complex, and K D, the dissociation constant, were estimated. The value of n was unity and K D was estimated to 146 nM. This K D value can be considered to be the association between the photo-intermediate and t-Htr, which is deduced by the method of estimation. Previously we (Photochem. Photobiol. 74 (2001) 489) reported a K D of 15 μM for the interaction between the wild-type and t-Htr by means of the change in M-decay rates. Therefore, this value should be the K D value for the interaction between M of the wild-type and t-Htr.

Effect of Cyclen on the Prolongation of M Intermediate Lifetime in the D96N Mutant of Bacteriorhodopsin

Abstract A new kind of chemical additive—cyclen (1,4,7,10-tetra-azacyclododecane) was used in BRD96N-PVA films in an attempt to obtain longer M state lifetime. At the highest cyclen concentration of 1:250, the lifetime of M state could be prolonged to 936 s. Comparing the lifetimes of the M state in BRD96N suspension and BRD96N-PVA film with the same cyclen content, it is the strong complexing properties of cyclen towards H+ that plays the most important role in this prolongation.