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1.
Hapten-linked conformational equilibria in immunolglobulins XRPC-24 and J-539 observed by chemical relaxation. 总被引:3,自引:0,他引:3
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The interaction of oligogalactan haptens with the murine myeloma proteins XRPC-24 and J-539 has been investigated by the fluorescence temperature-jump method. The relaxation spectrum is composed of two processes, the faster representing hapten assocaition and the slower a protein isomerization. In both cases the concentration dependence of relaxation times and amplitudes was consistent with the general mechanism formulated by Lancet and Pecht (1976, Proc. Natl. Acad. Sci. U.S.A. 73:3549), in which the equilibrium between two conformations of the protein is shifted by hapten binding. The intact proteins and their Fab fragment had identical kinetic behavior, indicating that the conformational changes are located in the Fab region. Temperature dependence analysis for protein J-539 permitted the calculation of activation parameters and led to a consistent energy profile for all the elementary steps. The conformational states are separated by large activation barriers, but have similar free energies. The results suggest that hapten-induced conformational changes in immunoglobulins are more general phenomena than was previously thought. 相似文献
2.
The combining site of the dinitrophenyl-binding immunoglobulin A myeloma protein MOPC 315 总被引:7,自引:6,他引:1
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Steven K. Dower Simon Wain-Hobson Peter Gettins David Givol W. Roland C. Jackson Stephen J. Perkins Christopher A. Sunderland Brian J. Sutton Carolyn E. Wright Raymond A. Dwek 《The Biochemical journal》1977,165(2):207-223
Magnetic-resonance techniques are used to refine the model of the combining site of the Fv fragment of the dinitrophenyl-binding mouse myeloma protein MOPC 315 constructed by Padlan, Davies, Pecht, Givol & Wright (1976) (Cold Spring Harbor Symp. Quant. Biol. 41, in the press). Light-absorption studies indicate a dinitrophenyl–tryptophan interaction in the Fv fragment of the type occurring in free solution. The Dnp-aspartate–tryptophan complex is therefore used as a starting point for the n.m.r. (nuclear-magnetic-resonance) analysis of the dinitrophenyl–Fv fragment interaction. Ring-current calculations are used to determine the geometry of the complex. The specificity of complex-formation between dinitrophenyl and tryptophan is confirmed by the lack of ring-current shifts of the dinitrophenyl resonances when tryptophan is replaced by any other aromatic amino acid. Proton n.m.r. difference spectra (at 270MHz), resulting from the addition of a variety of haptens to the Fv fragment, show that the combining site is highly aromatic in nature. Calculations on the basis of ring-current shifts define the geometry of the combining site, which involves a dinitrophenyl ring in van der Waals contact with four aromatic amino acid residues on the protein. The observation of a nuclear Overhauser effect on the H(3) resonance of the dinitrophenyl ring provides additional constraints on the relative geometry of the H(3) proton and an aromatic amino acid residue on the Fv fragment. The specificity of the Fv fragment for dinitrophenyl ligands arises from a stacking interaction of the dinitrophenyl ring with tryptophan-93L, in an `aromatic box' of essentially tryptophan-93L, phenylalanine-34H and tyrosine-34L; asparagine-36L and tyrosine-34L also contribute by forming hydrogen bonds with the nitro groups on the dinitrophenyl ring. The n.m.r. results also confirm that the antibody–hapten reaction may be visualized as a single encounter step. An Appendix shows the method of calculation of ring currents for the four aromatic amino acids and their use in calculating structures. 相似文献
3.
Specificity of interactions of hapten side chains with the combining site of the myeloma protein MOPC 315. 总被引:3,自引:3,他引:0
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S Wain-Hobson S K Dower P Gettins D Givol A C McLaughlin I Pecht C A Sunderland R A Dwek 《The Biochemical journal》1977,165(2):227-235
The pKa values of the three histidine residues in the Fv fragment (variable region of the heavy and light chains) of the mouse myeloma protein MOPC 315, measured by high resolution n.m.r. (nuclear magnetic resonance), are 5.9, 6.9 and 8.2. The perturbation of the pKa of one of the histidines (pKa 6.9) on the addition of hapten and the narrow linewidth of its proton resonances suggests that it is at the edge of the combining site. References to the model of the Fv fragment [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol. 41, in the press] allows assignment of the three histidine residues, histidine-102H, histidine-97L and histidine-44L. The determination of the pKa of the phosphorus group, by 31P n.m.r., of a homologous series of Dnp- and Tnp- (di- and tri-nitrophenyl) haptens has located a positively charged residue. Molecular-model studies on the conformations of these haptens show that the residue is at the edge of the site. The model suggests that the positively charged residue is either arginine-95L or lysine-52H. 相似文献
4.
The influence of the orientations of HO-2 and HO-3 on the puckering of the ring in (2S,3R)-tetrahydrofuran-2,3-diol (1) and (3R)-tetrahydrofuran-3-ol (2) has been investigated by consistent force-field methods. In the E or W regions of the pseudorotation cycle, there are no local energy minima. The local minima in the N and S regions are affected by different orientations of the hydroxyl groups, with up to 12.2 kJ.mol?1 for 1 and 6.9 kJ.mol?1 for 2. If statistical weights of different rotamers are used to calculate N/S equilibrium constants, both conformational states are equally populated. However, if the orientations of the hydroxyl groups are restricted to simulate behaviour of these molecular fragments in DNA double-helices, the equilibrium shifts towards N. The results indicate that, in the vapor state, a unique one-dimensional N-S energy-barrier does not exist and that there is a spectrum of barriers depending on the orientation of the hydroxyl groups. 相似文献
5.
The binding of 2,4,6-trinitrophenyl derivatives to the mouse myeloma immunoglobulin A protein MOPC 315 总被引:1,自引:1,他引:0
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Steven K. Dower Peter Gettins Roland Jackson Raymond A. Dwek David Givol 《The Biochemical journal》1978,169(1):179-188
The binding of Tnp (2,4,6-trinitrophenyl) derivatives to the Fv fragment (variable region of heavy and light chains) of the mouse myeloma IgA protein MOPC 315 was investigated by 270MHz proton nuclear magnetic resonance. Two of the haptens, Tnp-glycine and Tnp-l-aspartate, are in fast exchange with the Fv fragment, and the changes in chemical shifts for both protein and hapten resonances were determined by titrations. For the tightly binding hapten epsilon-N-Tnp-alpha-N-acetyl-l-lysine, which is in slow exchange with the Fv fragment, the changes in chemical shifts for the hapten H(3)+H(5) resonances were determined by cross-saturation. By using these data and the known structure of the combining site of protein MOPC 315 [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977), Nature (London) 266, 31-37] the mode of binding of Tnp derivatives is deduced by ring-current calculations. The trinitrophenyl ring stacks with tryptophan-93(L) (light chain) in the ;aromatic box' formed by tryptophan-93(L), tyrosine-34(L) and phenyl-alanine-34(H) (heavy chain). Further evidence for the stacking interaction with a tryptophan residue is provided by the similarity of the optical-difference spectra observed with Tnp-aminomethylphosphonate in the presence of either the Fab fragment (light chain and N-terminal half of heavy chain) of protein MOPC 315 or tryptophan. These data show that the modes of binding of all the Tnp derivatives are very similar, despite a 100-fold range in their affinities. It is also concluded that the modes of binding of Dnp (2,4-dinitrophenyl) and Tnp derivatives to protein MOPC 315 are very similar, and that the structural basis for this is that the aromatic box is large enought to allow the trinitrophenyl ring to stack with tryptophan-93(L) while still forming hydrogen bonds to asparagine-36(L) and tyrosine-34(L). 相似文献
6.
Toshiya Endo Masanao Oya Hiroshi Ozawa Yoshio Kawano José R. Giglio Tatsuo Miyazawa 《Journal of Protein Chemistry》1989,8(6):807-815
Proton nuclear magnetic resonance (NMR) spectra of crotamine, a myotoxic protein from a Brazilian rattlesnake (Crotalus durissus terrificus), have been analyzed. All the aromatic proton resonances have been assigned to amino acid types, and those from Tyr-1, Phe-12, and Phe-25 to the individual residues. ThepH dependence of the chemical shifts of the aromatic proton resonances indicates that Tyr-1 and one of the two histidines (His-5 or His-10) are in close proximity. A conformational transition takes place at acidicpH, together with immobilization of Met-28 and His-5 or His-10. Two sets of proton resonances have been observed for He-17 and His-5 or His-10, which suggests the presence of two structural states for the crotamine molecule in solution. 相似文献
7.
High-resolution, solid-state 13C-n.m.r. spectra were obtained for several crystalline cyclomaltohexaose inclusion-complexes. The resonances of C-1, C-4, and C-6 of the host were dispersed. The averaged 13C shifts of these resonances were in good agreement with the 13C shifts observed in solution, where the dispersion due to conformational diversity is expected to be averaged by rapid interconversion of the conformers. This result indicates that the most plausible source of the solid-state 13C-shift dispersions of the resonances of C-1 and C-4 is the diversity of conformations about the glycosidic linkage. The molecular origins of conformation-dependent 13C shifts are discussed. 相似文献
8.
Resonance Raman-spectroscopic studies of the hapten features involved in the binding of 2,4-dinitrophenyl haptens by the mouse myeloma proteins MOPC 315 and MOPC 460. 总被引:1,自引:1,他引:0
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The binding of four dinitrophenyl haptens to the mouse myeloma proteins MOPC 315 IgA (immunoglobulin A) and MOPC 460IgA was studied by resonance Raman spectroscopy. Isotopic substitution with 15N and 2H was used to assign features in the resonance Raman spectra of the free haptens. Changes in each of these features on binding to the proteins could then be attributed to interactions of the proteins' binding sites with either the p-NO2 or the o-NO2/amine regions of the haptens. The interactions between a given hapten and MOPC 315 IgA are often quite distinct from those between the same hapten and MOPC 460 IgA. Moreover, for both antibodies the nature of the R side chain in a Dnp-NHR (Dnp, 2,4-dinitrophenyl) compound appears to modify the interactions between the Dnp chromophore and the protein. Thus, with the haptens studied, there is no unique set of contacts between the Dnp group and the binding site. The contacts expected between epsilon-2,4-dinitrophenyl-L-lysine and the site on MOPC 315 IgA, on the basis of a recent model for this site [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977) Nature (London) 266, 31--37] were not detected. However, the contacts between this hapten and the site on MOPC 460 IgA were closer to those predicted by the model for MOPC 315 IgA. 相似文献
9.
We examine a short way to reach an exceptional point that corresponds to a coalescence of two resonance energies. The application concerns the photodissociation of the Na2 molecule exposed to a laser field. In this case, the resonances can be correlated with the field-free vibrational states of the diatomic species. The resonances are due to the field-induced coupling with the continuum of a repulsive potential. We also draw attention to a new kind of exceptional point involving a resonance originating from a vibrational state coalescing with a shape-type resonance of the repulsive potential. A laser control scenario, aiming at the adiabatic transport from this field-free decaying state to a stable field-free vibrational state, is discussed in terms of field-induced dissociation quenching. Figure
Laser-controlled Dissociation Quenching mechanism in Na2 using an Exceptional Point resulting from the merging of a shape-type resonance (R 8) and a Feshbach one originating from a vibrational bound state (v=0). The population transfer process is indicated by the blue arrow of the left panel. The laser-controlled energy trajectory from R 8 (decaying state) to v=0 (stable state) is displayed in the middle panel, while the survival probability is given in the right panel. An efficiency of about 25 % is reached for the quenching control. 相似文献
10.
R A Dwek R Jones D Marsh A C McLaughlin E M Press N C Price A I White 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》1975,272(915):53-74
This paper reports the initial progress in a research programme to identify and obtain the relative orientations, in solution, of the amino acid residues that constitute the combining site of the myeloma protein MOPC 315. This protein has a molecular mass of 150,000, but enzymic digestion yields the Fv fragment of molecular mass 25,000 which still has the combining site intact, as judged by the affinity for dinitrophenyl haptens. Analysis of the e.s.r. spectra of a series of dinitrophenyl spin labelled haptens has allowed the dimensions, rigidity and polarity profile of the combining site to be determined. The combining site is a cleft of overall dimensions 1.1 nm x 0.9 nm x 0.6 nm which has considerable structural rigidity. One of these spin labels has also been used to perturb the n.m.r. spectrum of the Fv and using difference spectroscopy the 270 MHz proton n.m.r. spectrum of the amino acid residues in and around the combining site has been obtained. This spectrum contains only the equivalent of about 30 aromatic and 21 aliphatic protons. Comparison of this difference spectrum with that obtained using a diamagnetic analogue suggests that any conformational changes on hapten binding are mainly localized to the combining site. By the use of (n.m.r.) difference spectroscopy the protons of the three histidine residues in the Fv are observed to titrate with pH and have pKa values of about 8.1, 6.9 and 6.1. The histidine resonances with pKa values 6.9 and 6.1 alter slightly in the presence of haptens and also appear in the spin label difference spectrum, and must therefore be in or near to the combining site. These are assigned to His 102H and His 97L. The existence of lanthanide binding sites on the Fv, necessary for the mapping studies, has been demonstrated by measurements of Gd III water relaxation rates in Fv solutions and also by the changes in the Fv tryptophan fluorescence on addition of Gd III. At pH 5.5 there is one tight binding site for the lanthanides (KD approximately 80 muM) but in the presence of hapten this is weakened 10-20 fold with a reciprocal effect on the hapten binding. Measurements of the Gd III quenching of the e.s.r. spectrum of a spin labelled hapten bound to Fv indicate that the lanthanide site is ca. 1.5 nm from the nitroxide moiety. 相似文献
11.
Shuji Yonezawa Kenichiro Fujiwara Takahiko Yamamoto Kazunari Hattori Hidekuni Yamakawa Chie Muto Motoko Hosono Yoshikazu Tanaka Toru Nakano Hiroshi Takemoto Mitsuhiro Arisawa Satoshi Shuto 《Bioorganic & medicinal chemistry》2013,21(21):6506-6522
For further investigation of BACE1 inhibitors using conformational restriction with sp3 hybridized carbon, we applied this approach to 6-substituted aminopyrimidone derivatives 3 to improve the inhibitory activity by reducing the entropic energy loss upon binding to BACE1. Among eight stereoisomers synthesized, [trans-(1′R,2′R),6S] isomer 6 exhibited the best BACE1 inhibitory activity, which was statistically superior to that of the corresponding ethylene linker compound (R)-3. Combinational examinations of the binding mode of 6 were performed, which included isothermal titration calorimetry (ITC), X-ray crystallographic structure analysis and theoretical calculations, to clarify the effect of our conformational restriction approach. From the ITC measurement, the binding entropy of 6 was found to be ~0.5 kcal larger than that of (R)-3, which is considered to be affected by conformational restriction with a cyclopropane ring. 相似文献
12.
The conformational properties of various oligonucleotides modified with the chemical carcinogen N-2-acetylaminofluorene have been investigated utilizing circular dichroism, proton magnetic resonance spectroscopy and computer-generated molecular models. Introduction of the carcinogen, specifically and covalently, at the C-8 position of guanosine residues results in dramatic changes in the ciruclar dichroism spectra of the oligonucleotides. The attachment of N-2-acetylaminofluorene also causes large higher-field shifts for the proton resonances of fluorene and bases adjacent to a modified guanosine residue. These results, together with substantial supporting evidence, show that the covalent binding of N-2-acetylaminofluorene causes important changes in the conformational properties of oligonucleotides in aqueous solution. The major changes include rotation of the guanine base around the glycosidic linkage and the intramolecular stacking of fluorene with an adjacent base. A computer-displayed model of a carcinogen-modified dinucleotide illustrating these effects is presented. The specific conformational changes noted for the oligonucleotides could clearly disrupt the normal biological activity of similarly modified naturally occurring nucleic acids. 相似文献
13.
The variability of nitro group--protein interaction in the 2,4-dinitrophenyl-binding antibodies M315, M460 and X25 investigated by resonance Raman spectroscopy.
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Pre-resonance Raman spectroscopy was used to study the interactions of the nitro groups of dinitrophenyl haptens with three dinitrophenyl-binding antibody fragments: M315 Fv, M460 Fab' and X25 Fab'. The observed changes in frequency of modes associated with the nitro moieties are compared with solvent-induced changes for the model hapten 2,4-dinitroaniline. These comparisons demonstrate a specific interaction via the H2N--C--C--2-NO2 and 4-NO2 groups with the protein. The interaction with the 4-NO2 group appears to be absent for epsilon-N-2,4-dinitrophenyl-L-lysine bound to M315 Fv fragment in contrast with either 2,4-dinitrophenylaspartate or 2,4-dinitrophenylglycine bound to M315 Fv fragment, despite the much tighter binding of the lysine derivative. The implications of this for M315 Fv fragment in terms of the antibody specificity are discussed. Comparisons of the effect of binding to M460 Fab' and X25 Fab' fragments also revealed significant differences in the shifts of the nitro group vibrations of 2,4-dinitrophenyl-lysine and 2,4-dinitroaniline. 相似文献
14.
《Carbohydrate research》1986,147(2):247-264
l-(1-13C, 5-2H)Arabinose (6D) and l-(2-13C, 5-2H)arabinose (8D) have been synthesized by degradation of 2,3-O-isopropylidene-α-l-rhamnofuranose (2) to l-(4-2H)erythrose (5β,5αD), with subsequent chain elongation to 6D plus l-(1-13C, 5-2H)ribose (7D), the latter being converted into 8D. Intermediates were identified by complete assignment of the 13C chemical shifts employing carbon-carbon and carbon-deuterium coupling constants, deuteration shifts, differential isotope-shifts, and deuterium spectra. The anomeric carbon atoms of 2 and 2,3-O-isopropylidene-l-(1-2H) erythrose (4D) gave only single 13C resonances, suggesting that these two compounds exists in only one major anomeric configuration, clarifying previously reported work. The synthesis of 2,3-O-isopropylidene-l-(1-2H)rhmanitol (3D) facilitated the assignment of the signals in the 13C spectra of the nondeuterated analog. Specific deuterium-enrichment and the observed carbon-deuterium coupling (1JC,D ∼22 Hz) not only served to identify the deuterated carbon atom unambiguously in 3 but also permitted assignment of closely spaced resonances. The deuterium spectrum of 2,3-O-isopropylidene-l-(1-2H)erythrofuranose (4D) showed only a single resonance, indicating preponderance of one anomer, in accord with the observation of a single C-1 resonance in the 13C spectrum. 相似文献
15.
Torrent J Marchal S Ribó M Vilanova M Georges C Dupont Y Lange R 《Biophysical journal》2008,94(10):4056-4065
Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique to trigger unfolding and refolding of wild-type ribonuclease A and a tryptophan-containing variant (Y115W). From the linear Arrhenius plots of the microscopic folding and unfolding rate constants, activation enthalpy (ΔH#), and activation entropy (ΔS#) were determined to characterize the kinetic transition states (TS) for the unfolding and refolding reactions. The single TS of the wild-type protein was split into three for the Y115W variant. Two of these transition states, TS1 and TS2, characterize a slow kinetic phase, and one, TS3, a fast phase. Heating T-jumps induced protein unfolding via TS2 and TS3; cooling T-jumps induced refolding via TS1 and TS3. The observed speed of the fast phase increased at lower temperature, due to a strongly negative ΔH# of the folding-rate constant. The results are consistent with a path-dependent protein folding/unfolding mechanism. TS1 and TS2 are likely to reflect X-Pro114 isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the β-hairpin comprising Trp115. A very fast protein folding/unfolding phase appears to precede both processes. The path dependence of the observed kinetics is suggestive of a rugged energy protein folding funnel. 相似文献
16.
Two series of dinitrophenyl haptens, in which chlorine replaces one or both nitro groups, were used to investigate, by a combination of high-resolution 1H n.m.r. and fluorescence quenching, the presence of groups in the combining site of protein MOPC 315, which form hydrogen bonds to the aromatic-ring substituents of the hapten. The large differences in binding constants on successive replacement of nitro groups were shown to be due to specific hapten-substituent-protein interactions by (a) showing that there was little difference in the interaction between these haptens and 3-methylindole (a model for the residue tryptophan-93L with which the hapten stacks in protein MOPC 315), (b) proving by 1H n.m.r. that the mode of hapten binding is constant and (c) showing that the differences in Kd were consistent with the relative hydrogen-bonding capacities of chlorine and the nitro moiety. In this way it was established that each nitro group forms a hydrogen bond. Furthermore, from consideration of the 1H n.m.r. chemical shifts of several dinitrophenyl haptens and their trinitrophenyl analogues, it was shown that there is no distortion of the o-nitro group on binding to the variable fragment of protein MOPC 315. 相似文献
17.
Böckmann A Lange A Galinier A Luca S Giraud N Juy M Heise H Montserret R Penin F Baldus M 《Journal of biomolecular NMR》2003,27(4):323-339
Solid state NMR sample preparation and resonance assignments of the U-[13C,15N] 2×10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra– and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all 15N, 13C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts. 相似文献
18.
Configurational assignments for the tertiary alcoholic centers of four branched-chain 3-C-nitromethylglycopyranosides, namely, methyl 2-benzamido-4,6-O-benzylidene-2-deoxy-3-C-nitromethyl-α-D-allopyranoside (1), benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-C-nitromethyl-α-D-glucopyranoside (4), benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-C-nitromethyl-α-D-allopyranoside (5), and methyl 4,6-O-benzylidene-3-C-nitromethyl-2-O-p-tolylsulfonyl-α-D-glucopyranoside (8), were made on the basis of the downfield chemical shifts of their identifiable protons per molar equivalent of added Eu(fod)3, as compared with those of model compounds, of known configuration, having a close structural relationship. In some cases, the assignments were corroborated by the position of the acetyl resonances in the unshifted 60-MHz p.m.r. spectra of the corresponding O-acetyl derivatives. 相似文献
19.
Interactions between a monoclonal, DNP-specific IgE molecules (hybridoma A2) and divalent DNP-haptens in solution cause aggregation of the former predominantly into closed rings of two IgE and two divalent haptens (Schweitzer-Stenner, R., A. Licht, I. Lüscher, and I. Pecht. 1987. Biochemistry. 26:3602-3612). The time course of this process was now investigated by titrating the A2-IgE with divalent DNP-haptens having long and rigid oligoproline spacers (di(N epsilon-2,4-dinitrophenyl)-6-amino-hexanoate-aspartyl-(prolyl)n-L-ly- syl; n = 24, 27, 33). Binding was expressed in quenching of the IgE intrinsic tryptophan emission. As shown in the preceding paper, hapten addition to the IgE-A2 at rates faster than a distinct threshold value led to nonequilibrium titrations (NETs) from which kinetic processes slower than 2 s-1 can be resolved. Analysis of these titrations shows that the dimeric rings open at rates of approximately 10(-2) s-1, independent of the divalent hapten's spacer length. The ring closure rate, however, decreases with spacer length. The latter observation was qualitatively rationalized in terms of the diffusion process of a Gaussian chain which relates the ring closure rate constant to the expectation value for the distance between the free ends of the respective open chain. 相似文献
20.
The adenosylcobalamin- (coenzyme B12) dependent ethanolamine ammonia-lyase (EAL) plays a key role in aminoethanol metabolism, associated with microbiome homeostasis and Salmonella- and Escherichia coli-induced disease conditions in the human gut. To gain molecular insight into these processes toward development of potential therapeutic targets, reactions of the cryotrapped (S)-2-aminopropanol substrate radical EAL from Salmonella typhimurium are addressed over a temperature (T) range of 220–250 K by using T-step reaction initiation and time-resolved, full-spectrum electron paramagnetic resonance spectroscopy. The observed substrate radical reaction kinetics are characterized by two pairs of biexponential processes: native decay to diamagnetic products and growth of a non-native radical species and Co(II) in cobalamin. The multicomponent low-T kinetics are simulated by using a minimal model, in which the substrate-radical macrostate, S?, is partitioned by a free-energy barrier into two sequential microstates: 1) S1?, a relatively high-entropy/high-enthalpy microstate with a protein configuration that captures the nascent substrate radical in the terminal step of radical-pair separation; and 2) S2?, a relatively low-enthalpy/low-entropy microstate with a protein configuration that enables the rearrangement reaction. The non-native, destructive reaction of S1? at T ≤ 250 K is caused by a prolonged lifetime in the substrate-radical capture state. Monotonic S? decay over 278–300 K indicates that the free-energy barrier to S1? and S2? interconversion is latent at physiological T-values. Overall, the low-temperature studies reveal two protein-configuration microstates and connecting protein-configurational transitions that specialize the S? macrostate for the dual functional roles of radical capture and rearrangement enabling. The identification of new, to our knowledge, intermediate states and specific protein-fluctuation contributions to the reaction coordinate represent an advance toward development of novel therapeutic targets in EAL. 相似文献