Conformational flexibility of the Cys 697-Cys 707 segment of myosin subfragment-1. Distance distributions by frequency-domain fluorometry

The separation between Cys 697 (SH1) and Cys 707 (SH2) of the heavy chain of myosin subfragment-1 was previously measured by fluorescence resonance energy transfer with a donor linked to SH1 and an acceptor to SH2. In the present study the distribution of the distances between the two thiols was recovered from frequency-domain fluorometry. In the native state and in the presence of ligands such as MgADP, pyrophosphate, orthovanadate (Vi) and actin, we found wide distributions of the separations between SH1 and SH2 (11-16 A) comparable to that found in the random-coil state (20 A). These results suggest that the SH1-SH2 segment has a high degree of conformational flexibility even in native S1. The flexibility is not much affected by the physiological state of S1. However, the ligands MgADP, Vi and MgADP + Vi decrease significantly the mean SH1-SH2 distance from 27 to 17 A with the effect of MgADP+ Vi being the most pronounced. The anisotropy decay of donor-labeled S1 is biphasic with two rotational correlation times. The long component is decreased by these ligands from 289 to 93 ns, suggesting a more compact symmetric structure of S1 in the presence of the ligands. The complex S1(MgADP)Vi has been shown to be a stable analogue of S1(MgADP)Pi, an unstable intermediate that is generated in the actomyosin ATPase cycle during muscle contraction. Since the power stroke of muscle is accompanied by release of Pi from S1(MgADP)Pi, the present results are consistent with a model in which force generation can be accompanied by transition of S1 from a highly symmetric or compact structure to a more extended structure.     

Ligand-induced myosin subfragment 1 global conformational change

The effects of selected ligands on the structure of myosin subfragment 1 (S1) were compared by using transient electrical birefringence techniques. With pairs of dilute solutions of S1 at 3.5 degrees C in low ionic strength (mu = 0.020 M) buffers that had matched electrical impedances, S1 with Mg2+, MgADP, or MgADP.Vi bound was subjected to 6-7-microseconds external electrical fields in the Kerr law range. Specific Kerr constants and the rates of rotational Brownian motion after the electric field was removed were measured. Neither Mg2+ nor MgADP had a measurable effect on either observable, but when orthovanadate (Vi) bound S1.MgADP it decreased the rotational correlation coefficient from 267 +/- 6 to 244 +/- 10 ns. Parallel measurements of MgATPase activity indicated that S1.MgADP.Vi was greater than 95% inhibited. These results confirm the conclusion of Aguirre et al. [(1989) Biochemistry 28, 799] that Vi binding to S1.MgADP increases its rate of rotational Brownian motion and provide data that are more quantitatively correlated with S1 structure. The Vi-induced change in the rotational correlation coefficient is consistent with S1 becoming more flexible or more compact when Vi binds. Assuming that S1.MgADP.Vi is an analogue for S1.MgADP.Pi, the structural changes observed for S1-ligand complexes in solution are discussed in relation to possible structural changes of intermediates on the kinetic pathway of ATPase hydrolysis. A new model of force generation by S1 in muscle is hypothesized.     

Limited trypsinolysis changes in structural dynamics of myosin subfragment 1

The effects of limited trypsinolysis of myosin subfragment 1 (S1) on its structural dynamics were investigated by using the method of transient electric birefringence. Conversion of S1 by trypsin to produce S1 (T) did not change the specific Kerr constant [(8.1 +/- 0.3) X 10(-7) and (8.0 +/- 0.3) X 10(-7) cm2/statvolt2 for S1(T) and S1, respectively] or the degree of alignment in a weak electric field, suggesting that the size of S1 and its permanent electric dipole moment are not modified by trypsin. On the other hand, the relaxation time for the field-free rotation, after achieving a steady-state birefringence signal, was reduced from 316 ns for S1 to 269 ns for S1(T), at 3.7 degrees C, suggesting that trypsinolysis increases the flexibility of the connections between S1 segments or introduces additional segmental motions. For both S1 and S1(T), the rate of decay for a steady-state signal was independent of the field strength, between 3.34 and 20.3 statvolt/cm. Shortening the duration of the weak electric field pulses to 0.35 microseconds, so that steady-state signals were not achieved, decreased the relaxation times for S1 and S1(T) to 240 and 210 ns, respectively, which is consistent with the segmented flexible S1 structure proposed earlier [Highsmith, S., & Eden, D. (1986) Biochemistry 25, 2237]. When the strength of the electric field was increased to above 10 statvolt/cm, in order to make the interaction energy for the S1(T) electric dipole moment in the electric field greater than the thermal energy, the relaxation time after a 0.35-microseconds pulse decreased from 210 to 170 ns as the field was increased from 7 to 20 statvolt/cm. (ABSTRACT TRUNCATED AT 250 WORDS)     

Anisotropic electric properties of purple membrane and their change during the photoreaction cycle

Purple membrane suspension shows two different orientations in electric fields of different frequencies. The orientation at low frequencies (less than or equal to approximately 10 Hz), with the membrane surface perpendicular to the electric field, is due to permanent dipole moment of the membrane and the orientation at high frequencies (greater than or equal to approximately 100 Hz), with the surface parallel to the electric field, is due to induced dipole moment. By quantitative analysis of these orientations, we determined the permanent dipole moment and the polarizability. Both values varied according to the membrane size: the permanent dipole moment ranged from 500 kD to 10 MD and was proportional to the square of the diameter of the membrane. The polarizability ranged from 1 X 10(-13) to 1 X 10(-11)cm3 and was proportional to the third to fourth power of the diameter. Because the permanent dipole moment was proportional to the area of the membrane, we could determine permanent dipole moment per bacteriorhodopsin. By determining the actual membrane size under electron microscopy, we got 98 D/bacteriorhodopsin. We also concluded that the direction of the permanent dipole moment was from the cytoplasmic to the extracellular side. These values, however, were strongly dependent on the ionic strength in the medium, suggesting a screening effect due to counter ions near the membrane surface. We evaluated the screening effect and showed about a four-charge difference between the two sides of the purple membrane. Under illumination, we found that the permanent dipole moment decreased from 98 to 63 D/bacteriorhodopsin. From the best-oriented sample, we also concluded that the angle of retinal against the axis normal to the membrane surface was greater than 68.6 degrees.     

Surface Charge Movements of Purple Membrane During Light-Dark Adaptation

The difference in the surface charge distribution between light-adapted and dark-adapted purple membranes was investigated with electric dichroism measurements from approximately pH 5 to pH 11. Purple membrane sheets in solution are oriented in a weak electric field by their permanent dipole moment, which is due to the charge distribution of the membrane surfaces and/or within the membrane. The degree of orientation of purple membrane sheets was obtained from the measurement of “electrical anisotropy” of retinal chromophore in the membranes. At about pH 7, there was no difference in the “electric anisotropy” between light- and dark-adapted purple membranes. At about pH 9, the electric anisotropy of dark-adapted purple membrane was larger than that of light-adapted purple membrane. But at around pH 6 the difference was opposite. Linear dichroism experiments did not show any change of retinal tilt angle with respect to the membrane normal between the two forms from approximately pH 5 to pH 10. This result indicates that the changes in the “electric anisotropy” are not due to the change of retinal tilt angle, but due to the change in the permanent dipole moment of the membrane. To estimate the change in surface charges from the permanent dipole moment, we investigated the difference of the permanent dipole moment between the native purple membrane and papain-treated purple membrane in which negative charges in the cytoplasmic-terminal part are removed. This estimation suggests that this light-dark difference at around pH 9 can be accounted for by a change of ~0.5 electric charge per bacteriorhodopsin (bR) molecule at either of the two surfaces of the membrane. We also found from pH electrode measurements that at about pH 8 or 9 light adaptation was accompanied by an uptake of ~0.1 protons per bR. A possible movement of protons during light-dark adaptation is discussed. The direction of the permanent dipole moment does not change with papain treatment. The permanent dipole moment in papain-treated purple membrane is estimated to be 27 ±2 debye/bR.     

Myosin subfragment 1 has tertiary structural domains

Transient electrical birefringence measurements were made on skeletal muscle myosin subfragment 1 (S1) at 3.7 degrees C in 10 mM tris(hydroxymethyl)aminomethane-acetate and 0.10 mM MgCl2, pH 7.0. The specific birefringence for 4.5 microM S1 was determined from steady-state measurements to be (8.1 +/- 0.3) X 10(-7) (cm/statvolt)2. For electric fields in the range of 2.47-24.7 statvolts/cm, the alignment was due to a large permanent dipole moment for S1, estimated to be 8500 +/- 2000 D. The duration and the strength of the transient electric field was varied, and the temporal response of the decay of the birefringence signal was analyzed. The rate of rotational motion after the field was removed increased with increasing field strength for short (0.35-microseconds) pulses and decreased with increasing pulse lengths for all field strengths. The rate of decay from a steady-state birefringence signal was independent of field strength. A model of S1 structure is proposed, which is consistent with these data and most other data on S1 structure. In this model, S1 is composed of two tertiary structural domains that are connected by a flexible linkage with a substantial restoring force. The electric dipole moments on the two domains are arranged head to tail. The segmental movement of the domains is restricted to certain directions. The average conformation of the molecule is elongated, but it can be made more compact by the torque exerted by an electric field. The structural changes depend on the strength and duration of the pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

UV-induced vanadate-dependent modification and cleavage of skeletal myosin subfragment 1 heavy chain. 1. Evidence for active site modification

Ultraviolet irradiation above 300 nm of the stable MgADP-orthovanadate (Vi)-myosin subfragment 1 (S1) complex resulted in covalent modification of the S1 and in the rapid release of trapped MgADP and Vi. This photomodified S1 had Ca2+ATPase activity 4-5-fold higher than that of the non-irradiated control S1, while the K+EDTA-ATPase activity was below 10% of controls. There was a linear correlation between the activation of the Ca2+ATPase and the release of both ADP and Vi with irradiation time. Analysis of the total number of thiols and the ability of photomodified S1 to retrap MgADP by cross-linking SH1 and SH2 with various bifunctional thiol reagents indicated that the photomodification did not involve these reactive thiols. Irradiation of the S1-MgADP-Vi complex caused a large increase in absorbance of the enzyme at 270 nm which was correlated with the release of Vi from the active site, suggesting an aromatic amino acid(s) was (were) involved. However, analysis by three different methods showed no loss of tryptophan. All the irradiation-dependent phenomena could be prevented by replacing Mg2+ with either Co2+, Mn2+, or Ni2+. Unlike previous irradiation studies of Vi-dynein complexes [Lee-Eiford, A., Ow, R. A., & Gibbons, I. R. (1986) J. Biol. Chem. 261, 2337-2342], no peptide bonds were cleaved in photomodified S1. Photomodified S1 was able to retrap MgADP-Vi at levels similar to unmodified S1. Upon irradiation of the photomodified S1-MgADP-Vi complex, MgADP and Vi were again released from the active site, resulting in heavy chain cleavage to form NH2-terminal 21-kDa and COOH-terminal 74-kDa peptides. All evidence indicates that this new photomodification and subsequent chain cleavage occur specifically at the active site.     

Electric birefringence of myosin subfragments.

Electric birefringence measurements have been made on aqueous solutions of myosin subfragments, heavy meromyosin, subfragments 1 and 2 (S-1 and S-2). All of these showed positive electric birefringence. Heavy meromyosin and S-2 showed a large intrinsic Kerr constant. From the analysis of the build up and decay process of the birefringence, the contribution of the slow induced dipole moment was concluded in heavy meromyosin and S-2, although the existence of the permanent dipole moment was not completely excluded. The decay process of the birefringence of heavy meromyosin was found to consist of two components; the fast one of which had a relaxation time of the same order as that of S-1. This is probably due to the presence of a flexible hinge in heavy meromyosin.     

Electric birefringence of myosin subfragments

Electric birefringence measurements have been made on aqueous solutions of myosin subfragments, heavy meromyosin, subfragments 1 and 2 (S-1 and S-2). All of these showed positive electric birefringence. Heavy meromyosin and S-2 showed a large intrinsic Kerr constant. From the analysis of the build up and decay process of the birefringence, the contribution of the slow induced dipole moment was concluded in heavy meromyosin and S-2, although the existence of the permanent dipole moment was not completely excluded. The decay process of the birefringence of heavy meromyosin was found to consist of two components; the fast one of which had a relaxation time of the same order as that of S-1. This is probably due to the presence of a flexible hinge in heavy meromyosin.     

The nature of protein dipole moments: experimental and calculated permanent dipole of alpha-chymotrypsin

The electric dichroism of alpha-chymotrypsin has been measured in buffers of various pH values and ion compositions. The stationary dichroism obtained as a function of the electric field strength is not compatible with an induced dipole mechanism and clearly shows that alpha-chymotrypsin is associated with a substantial permanent dipole moment. After correction for the internal directing electric field according to a sphere model, the dipole moment is 1.6 X 10(-27) C m at pH 8.3 (corresponding to 480 D). This value decreases with decreasing pH (to 1.2 X 10(-27) C m at pH 4.2), but is almost independent of the monovalent salt concentration in the range from 2 to 12 mM and of Mg2+ addition up to 1 mM. The assignment of the permanent dipole moment is confirmed by analysis of the dichroism rise curves. The dichroism decay time constants of (31 +/- 1) ns at 2 degrees C can be represented by a spherical model with a radius of 25-26 A, which is consistent with the known X-ray structure. The limiting linear dichroism is slightly dependent on the buffer composition and demonstrates subtle variations of the protein structure. As a complement to the experimental results, electric and hydrodynamic parameters of alpha-chymotrypsin have been calculated according to the known X-ray structure. Bead model simulations provide the center of diffusion, which is used to calculate dipole moments according to the equilibrium charge distribution evaluated from standard pK values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Linear contour length dependence of electrical polarizability of nucleosomal DNA fragments: implications for the flexibility of DNA

The transient electric birefringence of monodisperse oligonucleosomal DNA ranging from 145 to 990 base pairs has been studied. The orientation of fragments can be described in terms of an induced dipole moment with a small contribution of a permanent dipole. The electrical polarizability delta alpha was found to increase linearly with the DNA contour length. This unexpected dependence might result from a bent structure of DNA already considerable for very short segments. The observed delta alpha values agree with a segmental orientation of rigid subunits of length 13-18 nm as estimated in the elastic model of DNA with a kink angle of about 41 degrees.     

Electric birefringence studies on polyglutamic acid.

The electric birefringence for the aqueous solution of poly-L -glutamic acid (PGA) in the helical form was studied. PGA samples were fractionated by gel column chromatography. PGA showed a positive electric birefringence. The permanent dipole moment of the PGA molecule was suggested to be largely suppressed. The measurements of the intrinsic Kerr constants for various molecular lengths showed that the electric anisotropy (polarizability) of PGA is proportional to the 1.5 power of the length. The electric birefrigence measurement was also carried out in the helix–coil transition region. The Kerr constant of PGA was largely reduced on going from the helical form to the coiled form.     

Reversal of the surface charge asymmetry in purple membrane due to single amino acid substitutions.

Twenty-seven mutant bacteriorhodopsin's were screened to determine the PKa for reversal of the permanent electric dipole moment. The photoelectric response of an aqueous purple-membrane suspension was used to determine the direction of the purple-membrane dipole moment as a function of pH. The pK(a) for the dipole reversal of wild-type bacteriorhodopsin is 4.5. Six of the 27 mutant bacteriorhodopsin's were found to have a pK(a) for dipole reversal larger than that of wild-type bacteriorhodopsin. Two of these mutants, L93T and L93W, involve a neutral amino acid substitution in the interior of the protein. The direction of the purple-membrane permanent electric dipole moment is determined by the purple-membrane surface charge asymmetry. We conclude that these two substitutions, which do not involve charge replacement, alter the pK(a) for the reversal of the purple-membrane surface charge asymmetry. We suggest that these changes to the pK(a) are due to altered protein folding at the surface of the purple-membrane induced by single-site substitutions in the protein interior.     

Electrooptic study of the deionized form of bacteriorhodopsin

Electric field induced light scattering by suspensions of cation-depleted purple membranes, obtained by deionization of purple membrane (PM) suspensions on a cation exchange column or by electrodialysis at a pH around 6, shows a strong drop (more than 5 times) in the value of the permanent dipole moment relative to that of PM fragments. The membrane dipole moments were measured both at low dc and ac electric fields as well as by using electric field pulses with reversing polarity. Some slight changes in the dispersion of the electric polarizability were also observed.Microelectrophoretic measurements showed that the electric charge of the membrane fragments is increased by 30% after deionization. The importance of these data for the understanding of the blue membrane properties and subsequently for the mechanism of proton pumping are discussed.     

The radius of gyration of native and reductively methylated myosin subfragment-1 from neutron scattering.

Reductive methylation of nearly all lysine groups of myosin subfragment-1 (S1) was required for crystallization and solution of its structure at atomic resolution. Possible effects of such methylation on the radius of gyration of chicken skeletal muscle myosin S1 have been investigated by using small-angle neutron scattering. In addition, we have investigated the effect of MgADP.Vi, which is thought to produce an analog of the S1.ADP.Pi state, on the S1 radius of gyration. We find that although methylation of S1, with or without SO42- ion addition, does not significantly alter the structure, addition of ADP plus vanadate does decrease the radius of gyration significantly. The S1 crystal structure predicts a radius of gyration close to that measured here by neutron scattering. These results suggest that the overall shape by crystallography resembles nucleotide-free S1 in solution. In order to estimate the effect of residues missing from the crystal structure, the structure of missing loops was estimated by secondary-structure prediction methods. Calculations using the complete crystal structure show that a simple closure of the nucleotide cleft by a rigid-body torsional rotation of residues (172-180 to 670) around an axis running along the base of the cleft alone does not produce changes as large as seen here and in x-ray scattering results. On the other hand, a rigid body rotation of either the light-chain binding domain (767 to 843 plus light chains) or of a portion of 20-kDa peptide plus this domain (706 to 843 plus light chains) is more readily capable of producing such changes.     

紫膜碎片的电二色性研究

悬浮在水中的嗜盐菌紫膜碎片,在外电场作用下产主定向排列.在20℃时,568nm的电二色性研究表明:外加电场为2kV/m时取向程度可达60%以上;大于5.5kV/m时,取向作用趋于饱和状态;饱和时简约电二色性为-0.437左右,视黄醛生色团的跃迁矩方向与电偶极矩方向形成60.9°夹角;紫膜的永久偶极短为9.2×10~(-24)C、M,剩余电极化率为3.0×10~(-27)m~2;紫膜的旋转扩散常数为0.53秒~(-1).曲线拟合分析表明,感应偶极对紫膜碎片的定向的贡献应予考虑.本文对紫膜碎片的定向机理进行了讨论.     

Electrooptical measurements demonstrate a large permanent dipole moment associated with acetylcholinesterase.

Acetylcholinesterase (AChE) from krait (Bungarus fasciatus) venom is a soluble, nonamphiphilic monomer of 72 kDa. This snake venom AChE has been analyzed by measurements of the stationary and the transient electric dichroism at different field strengths. The stationary values of the dichroism are consistent with the orientation function for permanent dipoles and are not consistent with the orientation function for induced dipoles. The permanent dipole moment obtained by least-squares fits for a buffer containing 5 mM MES is 1000 D, after correction for the internal directing field, assuming a spherical shape of the protein. The dipole moment decreases with increasing buffer concentration to 880 D at 10 mM MES and 770 D at 20 mM MES. The dichroism decay time constant is 90 ns (+/- 10%) which is clearly larger than the value expected from the size/shape of the protein and indicates contributions from sugar residues attached to the protein. The dichroism rise times observed at low field strengths are larger than the decay times and, thus, support the assignment of a permanent dipole moment, although it has not been possible to approach the limit where the energy of the dipole in the electric field is sufficiently low compared to kT. The experimental value of the permanent dipole moment is similar to that calculated for a model structure of Bungarus fasciatus AChE, which has been constructed from its amino and acid sequence, in analogy to the crystal structure of AChE from Torpedo californica.     

Orientation of membrane fragments by electric field

Purple membrane fragments from Halobacterium halobium were oriented by a static electric field in a water suspension. It was found that an electric field of approx. 20 V/cm is sufficient to achieve practically complete orientation; the purple membranes have a permanent electric dipole moment of (6 ±1)· 10^?23 C · m, the orientation of the retinal transition moment relative to the direction of the electric dipole moment, θ, is (59 ± 1)⁰, and the purple membrane rotational diffusion constant D_rot = 0.65 s^?1. It was found that because of the electrophoretic movement of the particles a hydrodynamic velocity gradient builds up which also orients the purple membranes.     

Electric polarization of polyelectrolyte and colloid media: dielectric versus electro-optic approach

The theories of dielectric dispersion and of electric birefringence as a representative of electro-optic methods are considered and it is shown that they both depend in a similar way simply on the real part of the complex electric polarizability of the macromolecules or the particles. The latter also contains the permanent dipole moment. Experimental data on dielectric dispersion, electric birefringence and electric light scattering of strongly elongated, rod-like poly(tetrafluoroethylene) particles are compared and an attempt is made to extend the dielectric dispersion curve to lower frequencies using electric birefringence and electric light scattering data. Further, the experimental data on dielectric dispersion, electric light scattering, electro-orientation and dipolophoresis for the more complicated Escherichia coli particles are compared. Again, the possibility to extend the 10 kHz-100 MHz dielectric dispersion curve down below 1 Hz by using electric light scattering data is examined. The good matching of the dielectric dispersion and electric light scattering frequency curves found in the overlapping frequency range (10 kHz-5 MHz) essentially enhances the chance that dielectric dispersion below 1 MHz is related to alpha dispersion and not to electrode polarization. Thus it is not only possible to obtain additional information on the mechanism of polarization at lower-frequency dielectric dispersion, but also to extend our knowledge about the effective dielectric properties of biological complex fluids to frequencies essentially below 1 MHz. This could be important for the understanding of the effect of low-frequency electromagnetic fields on living matter.     

Electrostatics and electrodynamics of bacteriorhodopsin.

The stationary electric dichroism of bacteriorhodopsin is in qualitative, but not quantitative, agreement with the orientation function for disks having a permanent dipole directed perpendicular to the plane and an induced dipole in the plane. Fits of the orientation function to data measured at low field strengths demonstrate: an increase of the permanent dipole moment mu with the square of the disk radius r2, whereas the polarizability alpha increases with r4; the ionic strength dependence is small for mu and clearly stronger for alpha; the permanent dipole moment is 4x10(6) D at r = 0.5 micron. According to the risetime constants, the induced dipole does not saturate and increases to 4x10(8) D at 40 kV/cm and r = 0.5 micron. The data indicate that the permanent dipole is not of some interfacial character but is due to a real assymetry of the charge distribution. The experimental dipole moment per protein monomer is approximately 55 D, whereas calculations based on the structure of Grigorieff et al. (Grigorieff, N., T.A. Ceska, K.H. Downing, J.M. Baldwin, and R. Henderson. 1996. Electron-crystallographic refinement of the structure of bacteriorhodopsin. J. Mol. Biol. 259:393-421) provide a dipole moment of approximately 570 D. The difference is probably due to a nonsymmetric distribution of charged lipid residues. It is concluded that experimental dipole moments reflect the mu-potential at the plane of shear for rotational diffusion, in analogy to the sigma-potential used for translational diffusion. It is suggested that the permanent dipole of bacteriorhodopsin supports proton transport by attraction of protons inside and repulsion of protons outside of the cell. Dichroism rise curves at field strengths between E = 150 and 800 V/cm reveal an exponential component with time constants tau 3r in the range between 1 and 40 ms, which is not found in Brownian dynamics simulations on a disk structure using hydrodynamic and electric parameters characteristic of bacteriorhodopsin disks. The experimental data suggest that this process reflects a cooperative change of the bacteriorhodopsin structure, which is induced already at a remarkably low field strength of approximately 150 V/cm.