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PubMed2002

The SecYEG preprotein translocation channel is a conformationally dynamic and dimeric structure.

Escherichia coli preprotein translocase comprises a membrane-embedded trimeric complex of SecY, SecE and SecG. Previous studies have shown that this complex forms ring-like assemblies, which are thought to represent the preprotein translocation channel across the membrane. We have analyzed the functional state and the quaternary structure of the SecYEG translocase by employing cross-linking and blue native gel electrophoresis. The results show that the SecYEG monomer is a highly dynamic structure, spontaneously and reversibly associating into dimers. SecG-dependent tetramers and higher order SecYEG multimers can also exist in the membrane, but these structures form at high SecYEG concentration or upon overproduction of the complex only. The translocation process does not affect the oligomeric state of the translocase and arrested preproteins can be trapped with SecYEG or SecYE dimers. Dissociation of the dimer into a monomer by detergent induces release of the trapped preprotein. These results provide direct evidence that preproteins cross the bacterial membrane, associated with a translocation channel formed by a dimer of SecYEG.

Adenosine Triphosphatases↗
PubMed1995

The dependence of glucan conformational dynamics on linkage position and stereochemistry.

The 13C NMR T1 relaxation times for the (1-->4)-linked maltooligomers (Mi) and the (1-->6)-linked isomaltooligomers (IMi) with i = 2, 4, 6, and 8 were measured in aqueous solution at 22 and 65 degrees C at a concentration (3%) low enough to have removed concentration-dependent effects on the measured T1 values. Separate T1 values were measured for each carbon in the residue at the reducing end of the oligosaccharide, in the residue at the non-reducing end, and in the interior, i.e., non-terminal, residue(s). Analogous data for the corresponding high polymers show that at 22 degrees C the relaxation times for the carbons of the interior residues of the oligomers have converged to their high chain length asymptotes at about i = 10. This observation suggests that at room temperature polymeric motions in the frequency domain effective for 13C NMR relaxation at a magnetic field strength of 11.7 T have a "wavelength" of the order of 10 residues. The relaxation times characterizing the two ends of the chain are different, with longer T1 values for the carbons of the reducing end than for those of the non-reducing end. Carbons of alpha-anomeric residues at the reducing end have shorter relaxation times than those of the corresponding beta-anomeric reducing sugars. Carbons of the interior residues have T1 values shorter than the carbons of either type of terminal residue. For oligomers of a given dp there is no T1 difference between oligomers of the Mi and IMi series at room temperature. This observation is seemingly at odds with the great differences in the inherent conformational freedom of the (1-->4)- and (1-->6)-linkages. At elevated temperatures the orientational relaxation behavior of the two series of oligomers measured by 13C T1 values show interesting differences, and in the case of the Mi series, structure develops in the chain length dependence of the T1 values.

Carbohydrate Conformation↗
PubMed1985

Conformation, dynamics, and structural transitions of the TATA box region of self-complementary d[(C-G)n-T-A-T-A-(C-G)n] duplexes in solution.

Structural and kinetic features of the TATA box located in the center of the alternating self-complementary d(C-G-C-G-T-A-T-A-C-G-C-G) duplex (TATA 12-mer) and d(C-G-C-G-C-G-T-A-T-A-C-G-C-G-C-G) duplex (TATA 16-mer) have been probed by high-resolution proton and phosphorus NMR spectroscopy in aqueous solution. The imino exchangeable Watson-Crick protons and the nonexchangeable base protons in the TATA box of the TATA 12-mer and TATA 16-mer duplexes have been assigned from intra and inter base pair nuclear Overhauser effect (NOE) measurements. Imino proton line-width and hydrogen exchange saturation recovery measurements demonstrate that the dA X dT base pairs in the TATA box located in the center of the TATA 12-mer and TATA 16-mer duplexes are kinetically more labile than flanking dG X dC base pairs. The proton and phosphorus NMR parameters of the TATA 12-mer monitor a cooperative premelting transition in the TATA box prior to the onset of the melting transition to unstacked strands. Phosphorus NMR studies have been unable to detect any indication of a right-handed B DNA to a left-handed Z DNA transition for the TATA 12-mer duplex in saturated NaCl solution. By contrast, we do detect the onset of the B to Z transition for the TATA 16-mer in saturated NaCl solution. Proton and phosphorus NMR studies demonstrate formation of a loop conformation with chain reversal at the TATA segment for the TATA 12-mer and TATA 16-mer duplexes on lowering the DNA and counterion concentration. The imino protons (10-11 ppm) and phosphorus resonances (3.5-4.0 ppm; 4.5-5.0 ppm) of the loop segment fall in spectral windows well resolved from the corresponding markers in fully paired segments so tha it should be possible to identify loops in longer DNA helixes. The equilibrium between the loop and fully paired duplex conformations of the TATA 12-mer and TATA 16-mer is shifted toward the latter on addition of moderate salt.

Base Sequence↗
PubMed1979

On the structure and conformational dynamics of yeast phenylalanine-accepting transfer ribonucleic acid in solution.

The solution structure of yeast tRNAPhe was investigated by using ethidium as a fluorescent probe in the D loop and the anticodon loop. For this purpose the dihydrouracils in position 16/17 and wybutine in position 37 were substituted by ethidium. The lifetimes and the time-dependent anisotropy of ethidium fluorescence were measured by pulsed nanosecond fluorometry. The kinetics of the transitions between different states of the tRNAPheEtd derivatives were determined by chemical relaxation measurements. It was found that the ethidium label irrespective of its position exhibits three different states called T1, T2 and T3 characterized by lifetimes tau 1 = 30 ns, tau 2 = 12 ns, and tau 3 = 3 ns. The lifetime differences are due to different accessibilities of ethidium for solvent quenching in the three states. Thus, there are three different defined structural environments of the ethidium in both the anticodon and the D loop. The distribution of the three states was measured as a function of Mg2+ concentration and temperature; it was found that state T3 is favored over states T2 and T1 by both increasing Mg2+ concentration and increasing temperature. The chemical relaxation kinetics exhibit a fast transition between T1 and T2 (10--100 ms) and a slow transition between T2 and T3 (100--1000 ms). The rates of both transitions depend likewise on Mg2+ concentration and temperature. The equilibrium and kinetic data clearly show the presence of strong and weak interactions between Mg2+ and tRNA. A cooperative model accounting for this behavior is developed. The ethidium probe behaves identically when located in different regions of the tRNA regarding both its distribution of states and its transition kinetics. This suggests that the different spectroscopic states report different conformations of the tRNA structure. The dependence of the three states on Mg2+ and spermine indicates that conformation T3 is closely related to or identical with the crystal structure. The rotational diffusion constants indicate that of all three states T3 is most extended while T2 is most compact. The thermodynamic analysis reveals that the strongly bound Mg2+ ions reduce both the activation entropy and enthalpy of all transitions. The weakly bound Mg2+ ions increase both the activation enthalpy and entropy of the slow transition between T2 and T3. It is suggested that the breaking of several intramolecular bonds, e.g., hydrogen bonds, is involved in this transition.

Ethidium↗
PubMed2003

Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling.

S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10(-2)s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the "R1" residue reduced the k(cat) only for the labeled D107R1 form (7-fold). The K(m) value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K(m) for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by approximately 10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the viscosity dependence of the rate of AdoMet formation. The results suggest that the motion of the loop may be an intrinsic property of the protein and not be strictly ligand modulated.

Cysteine↗
PubMed2001

Conformational dynamics of a transposition repressor in modulating DNA binding.

The repressor of bacteriophage Mu functions in the establishment and maintenance of lysogeny by binding to Mu operator DNA to shut down transposition. A domain at its N terminus functions in DNA binding, and temperature-sensitive mutations in this domain can be suppressed by truncations at the C terminus. To understand the role of the C-terminal tail in DNA binding, a fluorescent probe was attached to the C terminus to examine its environment and its movement with respect to the DNA binding domain. The emission spectrum of this probe indicated that the C terminus was in a relatively hydrophobic environment, comparable to the environment of the probe attached within the DNA-binding domain. Fluorescence of two tryptophan residues located within the DNA-binding domain was quenched by the probe attached to the C terminus, indicating that the C terminus is in close proximity to this domain. Addition of DNA, even when it did not contain operator DNA, reduced quenching of tryptophan fluorescence, indicating that the tail moves away from the DNA-binding domain as it interacts with DNA. The presence of the tail also produced a trypsin hypersensitive site within the DNA-binding domain; mutant repressors with an altered or truncated C terminus were relatively resistant to cleavage at this site. Interaction of the wild-type repressor with DNA greatly reduced cleavage at the site. A repressor with a temperature-sensitive mutation in the DNA-binding domain was especially sensitive to cleavage by trypsin even in the presence of DNA, and the C-terminal tail failed to move in the presence of DNA at elevated temperatures. These results indicate that the tail sterically inhibits DNA binding and that it moves during establishment of repression. Such conformational changes are likely to be involved in communication between repressor protomers for cooperative DNA binding.

Amino Acid Sequence↗
PubMed1989

Conformational dynamics of individual DNA molecules during gel electrophoresis.

Gel electrophoresis is widely used in molecular biology to separate DNA molecules according to their sizes. The physical basis of this size separation is, however, poorly understood. Here we report observations of individual, fluorescently stained DNA molecules as they migrate during various kinds of gel electrophoresis. Their movement, under the influence of either a steady electric field or a pulsed-field, is characterized by cycles of elongation and contraction. Initially relaxed coils of DNA lengthen into 'hook-shaped' configurations which temporarily 'hang-up' on obstacles in the gel matrix before sliding off, contracting and entering another cycle. The effects of a new electrophoresis technique, termed 'pulse-oriented electrophoresis', which allows the effective angle of the electric field, and hence the molecular orientation of DNA, to be varied without electrode rearrangement, are also studied. In this case the DNA adopts a 'staircase' configuration showing that the net orientation in a direction is given by the vector sum of the pulses used.

DNA↗
PubMed1980

The excimer fluorescence of pyrene-labeled tropomyosin. A probe of conformational dynamics.

Rabbit skeletal and cardiac tropomyosin were specifically labeled at their cysteine side chains with N-(1-pyrene)-maleimide. A high degree of intramolecular excimer formation due to interaction between adjacent pyrenes on Cys 190 of each chain was observed by fluorescence techniques. This modification produced low values of specific viscosity at low salt, indicating an inhibition of the salt-dependent polymerizability. Despite the loss of polymerizability, the excimer fluorescence of pyrene-tropomyosin increased with a similar salt dependence as the decrease in viscosity of an unlabeled control. Transient and steady state fluorescence measurements on the labeled tropomyosin indicated the presence of two states of labeled tropomyosin, an excimer-forming state (State B) and a nonexcimer-forming state (State A), in equilibrium with each other. The increase in excimer with increasing temperature and salt concentration can be explained by a shift in equilibrium toward State B. Steric considerations suggest that, in order for the pyrenes to form an excimer, localized chain separation in State B is required.

Animals↗
PubMed2003

Conformational dynamics and temperature dependence of photoinduced electron transfer within self-assembled coproporphyrin:cytochrome c complexes.

The focus of the present study is to better understand the complex factors influencing intermolecular electron transfer (ET) in biological molecules using a model system involving free-base coproporphyrin (COP) complexed with horse heart cytochrome c (Cc). Coproporphyrin exhibits bathochromic shifts in both the Soret and visible absorption bands in the presence of Cc and an absorption difference titration reveals a 1:1 complex with an association constant of 2.63 +/- 0.05 x 10(5) M(-1). At 20 degrees C, analysis of time-resolved fluorescence data reveals two lifetime components consisting of a discrete lifetime at 15.0 ns (free COP) and a Gaussian distribution of lifetimes centered at 2.8 ns (representing (1)COP --> Cc ET). Temperature-dependent, time-resolved fluorescence data demonstrate a shift in singlet lifetime as well as changes in the distribution width (associated with the complex). By fitting these data to semiclassical Marcus theory, the reorganizational energy (lambda) of the singlet state electron transfer was calculated to be 0.89 eV, consistent with values for other porphyrin/Cc intermolecular ET reactions. Using nanosecond transient absorption spectroscopy the temperature dependences of the forward and thermal back ET originating from triplet state were examined ((3)COP --> Cc ET). Fits of the temperature dependence of the rate constants to semiclassical Marcus theory gave lambda of 0.39 eV and 0.11 eV for the forward and back triplet ET, respectively (k(f) = (7.6 +/- 0.3) x 10(6) s(-1), k(b) = (2.4 +/- 0.3) x 10(5) s(-1)). The differing values of lambda for the forward and back triplet ET demonstrate that these ET reactions do not occur within a static complex. Comparing these results with previous studies of the uroporphyrin:Cc and tetrakis (4-carboxyphenyl)porphyrin:Cc complexes suggests that side-chain flexibility gives rise to the conformational distributions in the (1)COP --> Cc ET whereas differences in overall porphyrin charge regulates gating of the back ET reaction (reduced Cc --> COP(+)).

Animals↗
PubMed1995

Conformational dynamics monitored by His-179 and His-200 of isolated thermophilic F1-ATPase beta subunit which reside at the entrance of the 'conical tunnel' in holoenzyme.

When monitored by 1H NMR at various pH values, most of the C-2 proton signals from 12 His residues of the isolated beta subunit of thermophilic F1-ATPase (TF1) could be separately observed. Two of them were assigned to His-179 and His-200 which reside at the entrance of a 'conical tunnel' to reach catalytic site in the crystal structure of F1-ATPase. His-200 gave doublet, suggesting that this region is not a rigid alpha-helix in the isolated beta subunit. The binding of Mg.AMP-PNP changed the chemical shifts of His-179 and His-200 significantly. Although His-119 located at the opposite side of the conical tunnel was not affected by the nucleotide-binding, it contributed to the stability of beta subunit and the efficiency of the catalysis of the holoenzyme.

Bacillus↗
PubMed1990

Conformational dynamics in fluorophenylcarbamoyl-alpha-chymotrypsins.

A series of fluorine-substituted diphenylcarbamoyl chlorides have been synthesized and used to prepare corresponding diphenylcarbamoylated derivatives of alpha-chymotrypsin. The enzyme is rapidly inactivated by these compounds, as has been previously observed for the unsubstituted chloride, and the derivatives are stable enough to permit extensive studies by fluorine NMR spectroscopy. In combination with previously reported results, these NMR experiments suggest that the aromatic rings of a diphenylcarbamoyl group attached to chymotrypsin may be found in two magnetically and dynamically distinguishable sites, with exchange between these sites taking place by a process that involves rotation about the carbamoyl N-CO bond and localized unfolding of the enzyme. The extent to which a given fluoroaromatic ring is found in one of these sites is dependent on the position of the fluorine substituent and the nature of the partner aromatic ring. It is found that a 2-fluorophenyl ring, when present, dominantly determines site occupation, while a 3-fluorophenyl ring has no effects that are detectably different from those of an unsubstituted phenyl ring. There is evidence for slow aromatic ring rotation within at least one of the phenyl ring interaction sites. Saturation transfer and lineshape methods provide information about the rates of interconversion of the N-phenyl groups between these sites. Line-width, spin-lattice relaxation times and fluorine-proton nuclear Overhauser effects determined at 282 and 470 MHz are reported for each system examined.

Binding Sites↗
PubMed1991

Conformational dynamics and solvent viscosity effects in carboxypeptidase-A-catalyzed benzoylglycylphenyllactate hydrolysis.

We have used a new approach to the dynamics of hydrolytic metalloenzyme catalysis based on investigations of both external solvent viscosity effects and kinetic 2H isotope effects. The former reflects solvent and protein dynamics, and the nuclear reorganization distribution among damped protein motion and intramolecular friction-free nuclear motion. The isotope effect represents proton tunnelling and reorganization in the hydrogen bond network around the active site. We illustrate the approach by new spectrophotometric and pH-titration data for carboxypeptidase-A-catalyzed benzoylglycyl-L-phenyllactate hydrolysis. This substrate exhibits both a significant inverse fractional power law viscosity dependence over wide ranges controlled by glycerol and sucrose, and a kinetic 2H isotope effect of 1.65. The analogous benzoylglycylphenylalanine hydrolysis has a smaller isotope effect (1.3) and no viscosity dependence. Viscosity variation has no effect on the CD spectra in the 180-240-nm range. In terms of stochastic chemical rate theory, the data correspond to an enzyme-peptide substrate complex with a 'tight' structure protected from the solvent. In comparison, the enzyme-ester substrate complex is 'softer', strongly coupled to the solvent, and the rate-determining step is accompanied by proton transfer or by substantial reorganization in the hydrogen bonds near the active site.

Carboxypeptidases↗
PubMed1979

Proton N.M.R. study of the conformational dynamics of porcine pancreatic colipase. Titration of aromatic residues.

The low-field portion of the 360 MHz proton N.M.R. spectrum of native porcine pancreatic colipase has been studied as a function of pH over the pH range 2-12. Resonances associated with the 26 protons of the aromatic rings of the two histidines, two phenylalanines and three tyrosines have been identified and tentatively assigned to specific residues. Titrations of pH yielded apparent pKa's of 7.9, 6.9, 10.4, 10.3 and 11.3 for His I (His 30), His II (His 86), Tyr I (Tyr 56 or 57), Tyr II (Tyr 56 or 57) and Tyr III (Tyr 53) respectively (tentative assignments). The high pKa value of His 30 is attributed to the vicinity of Asp 31. The mobility of the aromatic ring of Tyr 53 is hindered and an upper bound of 500 s-1 on the rate of rotation can be estimated. The aromatic rings of the 2 other tyrosine residues and of the 2 phenylalanine residues can rotate freely on the N.M.R. time scale. The study of perturbations in titration profiles and chemical shift values reveals a specific interaction of His 86 with Tyr I and, to a lesser extent, Tyr II. The existence of this interaction indicates that the protein folding brings in close spatial vicinity two distant regions of the covalent structure to form a "hydrophobic-aromatic" site which might be involved in the binding of bile salt micelles to pancreatic colipase.

Amino Acid Sequence↗
PubMed1994

Evidence for conformational dynamics and molecular aggregation in cytochrome P450 102 (BM-3).

The native molecular weight of affinity-purified cytochrome P450 102 from barbiturate-induced Bacillus megaterium has been studied by sedimentation methods and HPLC size-exclusion chromatography. Sedimentation velocity experiments yielded an s020,w = 9.244 S for the holocytochrome, but the diffusion coefficient was unexpectedly large and varied widely with centrifugal field, ionic strength, and protein concentration. Addition of 50 mM DL-dithiothreitol (DTT) caused a small decrease in the value of s020,w, but D20 still did not behave as expected. The sedimentation coefficients were consistent with a molecular weight of about 200,000, and the diffusion coefficients indicated molecular aggregation. Sedimentation equilibrium analyses suggested that the native enzyme was a mixture of monomer, dimer, trimer, and tetramer. However, after incubation of P450 102 with DTT, sedimentation equilibrium demonstrated that the enzyme was dimeric (molecular weight 236,000). HPLC size-exclusion chromatography of the cytochrome showed the presence of four peaks, which corresponded to 1.45-mer, 2.06-mer, 3.02-mer, and a higher molecular weight fraction; aggregated forms accounted for about 52% of the P450 102. Incubation of the enzyme with DTT caused a shift toward the 1.45-mer, but dimer, trimer, and the high molecular weight peak still persisted; the shift was not attributable to disulfide bond reduction. The 1.45-mer was determined to be a monomeric species of significantly asymmetric geometry. Together, the results indicated that cytochrome P450 exists with monomer, dimer, trimer, etc. in equilibrium, contrary to the expectation that this soluble P450 would be monomeric.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus megaterium↗
PubMed1997

Conformational dynamics and kinetics of peptide antagonist interactions with interleukin-1 receptor. Fluorescence studies using the NBD-labelled peptide AF12415.

Interleukin-1 plays a key role in the inflammatory response provoked by various disease states and inhibition of its action can bring therapeutic benefits. Steady-state and time-resolved studies of the intrinsic tryptophan fluorescence of the free soluble Type I form of interleukin-1 receptor (IL-1R) reveal that the rotational motions of the three major domains are strongly associated. Bound peptide antagonists are buried in hydrophobic regions, but a flexible association permits access to species from the aqueous phase. Ligand binding does not lead to rigidification of the receptor structure. The kinetics and mechanism of complex formation and dissociation, involving IL-1R with receptor antagonist protein (IL-1ra) and with peptides AF11733 (15 aa) and AF10961 (21 aa) were determined with the aid of peptide AF12415 (15 aa) labeled at its N-terminus by the NBD fluorophore, which exhibits a five-fold increase in emission intensity at 540 nm on binding of the peptide to IL-1R. The magnitude of the ON rate constant, typically 1 x 10(6) M-1 s-1, implies the existence of an intermediate 'encounter complex' involving interactions of low specificity. Readjustments of the initial encounter complex leads to a final complex where very specific interactions dominate. The first-order rate constant for this latter process is the most sensitive indicator of the true peptide affinity for the receptor binding site, and thus provides a better criterion than the apparent OFF rate (typically 2 x 10(-3) s-1) for discrimination of peptide antagonists.

Amino Acid Sequence↗
PubMed1989

Fluorescence studies of the conformational dynamics of parvalbumin in solution: lifetime and rotational motions of the single tryptophan residue.

The fluorescence properties of the single tryptophan residue in whiting parvalbumin were used to probe the dynamics of the protein matrix. Ca2+ binding caused a blue-shift in the emission (from lambda max = 339 to 315 nm) and a 2.5-fold increase in quantum yield. The fluorescence decay was nonexponential in both Ca2(+)-free and Ca2(+)-bound parvalbumin and was best described by Lorentzian lifetime distributions centered around two components: a major long-lived component at 2-5 ns and a small subnanosecond component. Raising the temperature from 8 to 45 degrees C resulted in a decrease in both the center (average) and width (dispersion) of the major lifetime distribution component, whereas the center, width, and fractional intensity of the fast component increased with temperature. Arrhenius activation energies of 1.3 and 0.3 kcal/mol were obtained in the absence and in the presence of Ca2+, respectively, from the temperature dependence of the center of the major lifetime distribution component. Direct anisotropy decay measurements of local tryptophan rotations yielded an activation energy of 2.3 kcal/mol in Ca2(+)-depleted parvalbumin and indicated a correlation between rotational rates and lifetime distribution parameters (center and width). Ca2+ binding produced a decrease in the width of the major lifetime distribution component and a decrease in tryptophan rotational mobility within the protein. There was a rough correlation between these two parameters with changes in Ca2+ and temperature, so that both measurements may be taken to indicate that the structure of Ca2(+)-bound parvalbumin was more rigid than in Ca2(+)-depleted parvalbumin.

Calcium↗
PubMed1979

Fluorescence studies on lipoamide dehydrogenases of pig heart. I. Conformational dynamics of enzyme.

The dynamic structures of two major forms (LD(I) and LD(II) of pig heart lipoamide dehydrogenase, resolved by TEAE-cellulose column chromatography, were studied by fluorescence depolarization. FAD and ANM were used as an intrinsic and an extrinsic fluorescent probe, respectively. In the experiments with bound FAD of lipoamide dehydrogenase, no thermal dependence of the fluorescence depolarization of either enzyme was observed and the values of polarization were close to the theoretical maximum value of 0.5. Both enzymes contained two reactive thiol groups which differed in their reactivities toward ANM. When the enzymes were labeled with one mol of ANM per mol of enzyme, the rotational relaxation times of LD(I) and LD(II) were found to be 18 ns and 196 ns, respectively. These findings indicate that the sement of LD(I) labeled with ANM fluctuates in the order of nanoseconds, whereas this segment of LD(II) is fixed rigidly. On the other hand, when the enzymes were labeled with two mol of ANM per mol of enzyme, both enzymes showed the composite result of fluorescence depolarization due to the motilities of the segment of enzyme and the whole enzyme molecule. These findings indicate that both LD(I) and LD(II) have the non-equivalent motilities of segments containing one reactive thiol group between the two monomers. In other words, the segment containing the ANM binding site of the one monomer is flexible and this segment of the other monomer is fixed rigidly in both enzymes.

Animals↗
PubMed1996

Rotational and conformational dynamics of Escherichia coli ribosomal protein L7/L12.

Fluorescence methods were utilized to study dynamic aspects of the 24 kDa dimeric Escherichia coli ribosomal protein L7/L12. Oligonucleotide site-directed mutagenesis was used to introduce cysteine residues at specific locations along the peptide chain, in both the C-terminal and N-terminal domains, and various sulfhydryl reactive fluorescence probes (iodoacetamido) fluorescein, IAEDANS, pyrenemethyl iodoacetate) were attached to these residues. In addition to the full-length proteins, a hinge-deleted variant and variants corresponding to the C-terminal fragment and the N-terminal fragment were also studied. Both steady-state and time-resolved fluorescence measurements were carried out, and the results demonstrated that L7/L12 is not a rigid molecule. Specifically, the two C-terminal domains move freely with respect to one another and with respect to the dimeric N-terminal domain. Removal of the hinge region, however, significantly reduces the mobility of the C-terminal domains. The data also show that the rotational relaxation time monitored by the fluorescent probe-depends upon the probe's excited state lifetime. This observation is interpreted to indicate that a hierarchy of motions exists in the L7/L12 molecule including facile motions of the C-terminal domains and dimeric N-terminal domain, in addition to the overall tumbling of the protein. Probes attached to the N-terminal domain exhibit global rotational relaxation times consistent with the molecular mass of the dimeric N-terminal fragment. Upon reconstitution of labeled L7/L12 with ribosomal cores, however, the motion associated with the dimeric N-terminal domain is greatly diminished while the facile motion of the C-terminal domains is almost unchanged.

Bacterial Proteins↗