共查询到20条相似文献,搜索用时 15 毫秒
1.
Yin J Beuscher AE Andryski SE Stevens RC Schultz PG 《Journal of molecular biology》2003,330(4):651-656
The germline precursor to the ferrochelatase antibody 7G12 was found to bind the polyether jeffamine in addition to its cognate hapten N-methylmesoporphyrin. A comparison of the X-ray crystal structures of the ligand-free germline Fab and its complex with either hapten or jeffamine reveals that the germline antibody undergoes significant conformational changes upon the binding of these two structurally distinct ligands, which lead to increased antibody-ligand complementarity. The five somatic mutations introduced during affinity maturation lead to enhanced binding affinity for hapten and a loss in affinity for jeffamine. Moreover, a comparison of the crystal structures of the germline and affinity-matured antibodies reveals that somatic mutations not only fix the optimal binding site conformation for the hapten, but also introduce interactions that interfere with the binding of non-hapten molecules. The structural plasticity of this germline antibody and the structural effects of the somatic mutations that result in enhanced affinity and specificity for hapten likely represent general mechanisms used by the immune response, and perhaps primitive proteins, to evolve high affinity, selective receptors for so many distinct chemical structures. 相似文献
2.
To reveal non-covalent interactions between the Fab and Fc regions of IgG molecules the average conformational free-energy change (delta Go), associated with reversible micro-unfoldings, was measured by hydrogen-deuterium exchange for the Fab and Fc fragments and the complete molecule. Human monoclonal IgG1 and pooled IgG samples were used in these experiments. Hydrogen-deuterium exchange data were summarized and compared in the form of exchange relaxation spectra. The experimentally observed relaxation spectrum of intact IgG could not be deduced by weighted summation of spectra measured for Fab and Fc fragments. A comparison of the measured and calculated data revealed a 5-kJ/mol increase in the conformational free energy upon splitting the IgG molecule into two Fab and Fc pieces, i.e. an increase of conformational mobility occurred. This change can be explained either by related fluctuation patterns of the Fab and Fc pieces in the intact molecule or by a shielding effect on the contact surfaces. Both interpretations suppose non-covalent interactions between Fab and Fc that can be a means of information transduction between recognition and effector sites. The pH dependence of the hydrogen-deuterium exchange also indicates interactions between the Fab and Fc regions. A shift in the relaxation spectra of the Fab fragment was observed between pH 8.2 and 7.3 revealing destabilization of the structure at lower pH. This effect is absent in the intact molecule, reflecting interactions that stabilize the Fab structure. Comparison of the relaxation spectra of Fab and Fc shows a difference of about 10 kJ/mol in the microstability of these fragments: the Fab part possesses more conformational flexibility (i.e. its microstability is smaller) than the Fc part. 相似文献
3.
Two conformational states of the coat protein of the filamentous bacteriophage M13 have been detected in detergent solution by using magnetic resonance techniques. When 3-fluorotyrosine is incorporated in place of the two tyrosine residues in the protein, four 19F nuclear magnetic resonance signals are observed, two for each conformer of the protein. The equilibrium between the two forms can be modulated by pH, temperature, and detergent structure. The rate of interconversion of the isomers is rapid on the minutes time scale but is slow relative to the T1 relaxation time of the fluorine resonances of approximately 50 ms. The conformational change between the conformers results in the perturbation of a basic residue in the protein such that this group has a pKa of approximately 9.5 in one state which shifts to 10.5 or more in the other conformational state. The temperature dependence of the equilibrium suggests an enthalpy difference of about 10 kcal/mol which is offset by entropy to give nearly zero free energy difference between the states at pH 8.3 in deoxycholate solution at room temperature. This suggests a substantial reorganization of the noncovalent interactions defining the two conformational states. The conformational equilibrium is strongly dependent on detergent structure and the presence of phospholipid in the detergent micelle. The results are not consistent with a strong, specific lipid binding to the protein but appear to be consistent with a more general effect of the overall micelle structure on the conformational state of the protein. 相似文献
4.
When a protein's active site happens to be strongly coupled with the protein structure, the rate constant of the reaction may eventually be modulated by the conformational fluctuations. Evidence for this effect has long been provided by extensive flash photolysis investigations of liganded hemoproteins and more recently of the non-heme respiratory protein hemerythrin in hydro-organic solvents. Within a given protein conformational substate, an elementary reaction step is characterized by one single free energy barrier and by a first-order rate constant, k, which changes with temperature according to an Arrhenius law. At physiological temperature and low viscosity, ultrafast conformational relaxation causes efficient averaging of the reaction rates and the protein displays exponential kinetics with an average rate constant (k). Under sufficiently general conditions, it can be shown that (k) also follows a simple Arrhenius law with 'effective' values of the pre-exponential factor Aeff and activation enthalpy Heff. It is found that Aeff strongly depends on the overall shape of the rate constant distribution and that Heff actually corresponds to the lower limit of the enthalpy of activation, i.e. the value associated with the highest possible reaction rate. The underlying distribution of rate constants can be reconstructed from a set of experiments in which the kinetics depart from an exponential, i.e. at low temperature and high viscosity. The most probable distribution of exponentials consistent with the observed kinetics of the geminate recombinations of oxygen with photodissociated hemerythrin has been determined by using a new approach, known as the maximum entropy method. The results are consistent with a single pre-exponential value and a distributed enthalpy spectrum. As expected, Heff does not coincide either with the most probable nor with the average value of the enthalpy. The most salient findings are that the probability for any protein molecule to have an enthalpy of activation equal to the effective value Heff vanishes and that Aeff differs by nearly three orders of magnitude from the true value A0. Biochemical reaction rates are actually average values, since protein reactions are measured under physiological conditions, where conformational relaxation is always fast. Our understanding of the significance of Aeff and Heff is therefore entirely dependent on the knowledge of the distribution function of the rate constants. In particular, enthalpy and entropy terms of similar reactions performed by different proteins cannot be compared as long as the distribution of the rate constants remains unknown. 相似文献
5.
This study presents circular dichroism (CD) spectra of a high-affinity monoclonal anti-fluorescein antibody (Mab 4-4-20), its Fab fragments, and corresponding single-chain antibody (SCA). In the region 200-250 nm, the differences in the CD spectra between these proteins reflect the uneven distribution of chromophores (tryptophan and tyrosine) rather than a major conformational change. On the basis of near-UV CD spectra, binding of the hapten fluorescein to these protein antibodies elicits an increased asymmetry in the microenvironment of the chromophoric residues in contact with the hapten and also perturbs the interface between VL and VH domains. The hapten-binding site provides a chiral microenvironment for fluorescein that elicits a pronounced induced fluorescein CD spectrum in both the visible and UV regions. In contrast to the parent molecules, SCA is thermolabile. Our results demonstrate that (1) UV CD spectra are useful for assessing the chromophoric microenvironment in the binding portion of antibodies and (2) the extrinsic fluorescein hapten CD spectra provide information about the interaction of hapten with the binding pocket. 相似文献
6.
The conformation of two fragments derived from anti-poly(D-alanyl) antibodies, the divalent fragment (Fab')2 and the monovalent fragment Fab', was studied by small-angle X-ray scattering before and after interaction with the tetra-D-alanine amide hapten. More than 90% of the combining sites were occupied by the hapten. No significant changes were observed in the volume or in the radius of gyration, with either of the fragments. This contrasts with the significant decrease in these two parameters found upon reacting the hapten with intact anti-poly(D-alanyl) antibodies (I. Pilz, O. Kratky, A. Licht, and M. Sela (1973), Biochemistry 12, 4998). For Fab', the radius of the whole particle was found to be 3.48 nm in the absence of the hapten and 3.46 nm in its presence, the radius of gyration of the cross-section was 1.37 nm without hapten and 1.38 nm in its presence, and the volume of the particle was 98 nm3 in the absence of the hapten and 91 nm3 in its presence. For (Fab')2 the respective values were 5.06 and 5.05, 1.38 and 1.37, and 182 and 182. These results suggest that a conformational change occurs within the antibody molecule, but not within its Fab fragment, upon reaction with the tetraalanine hapten. 相似文献
7.
Bacteriorhodopsin of halobacterial purple membranes exhibits conformational flexibility in high electric field pulses (1-30 x 10(5) V m(-1), 1-100 micros). High-field electric dichroism data of purple membrane suspensions indicate two kinetically different structural transitions within the protein; involving a rapid (approximately 1 micros) concerted change in the orientation of both retinal and tyrosine and/or tryptophan side chains concomitant with alterations in the local protein environment of these chromophores. as well as slower changes (approximately 100 micros) of the microenvironment of aromatic amino acid residues concomitant with pK changes in at least two types of proton-binding sites. Light scattering data are consistent with the maintenance of the random distribution of the membrane discs within the short duration of the applied electric fields. The kinetics of the electro-optic signals and the steep dependence of the relaxation amplitudes on the electric field strength suggest a saturable induced-dipole mechanism and a rather large reaction dipole moment of 1.1 x 10(-25) C m ( = 3.3 x 10(4) debye) per cooperative unit at E = 1.3 x 10(5) V m(-1), which is indicative of appreciable cooperativity in the probably unidirectional transversal displacement of ionic groups on the surfaces of and within the bacteriorhodopsin proteins of the membrane lattice. The electro-optic data of bacteriorhodopsin are suggestive of a possibly general, induced-dipole mechanism for electric field-dependent structural changes in membrane transport proteins such as the gating proteins in excitable membranes or the ATP synthetases. 相似文献
8.
Yin J Mundorff EC Yang PL Wendt KU Hanway D Stevens RC Schultz PG 《Biochemistry》2001,40(36):10764-10773
In an effort to gain greater insight into the evolution of the redox active, catalytic antibody 28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, and the X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 were determined. Comparison with the previously determined structures of the unliganded and hapten-bound affinity-matured Fab [Hsieh-Wilson, L. C., Schultz, P. G., and Stevens, R. C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 5363] shows that the germline antibody binds the p-nitrophenyl ring of hapten 3 in an orientation significantly different from that seen in the affinity-matured antibody, whereas the phosphonate moiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has more electrostatic and hydrophobic interactions with hapten 3 than the germline antibody and binds the hapten in a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3 and CDR L1 upon hapten binding to the germline antibody, consistent with the notion of structural plasticity in the germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G., and Stevens, R. C. (1997) Science 276, 1665]. The structural differences are reflected in the differential binding affinities of the germline Fab (K(d) = 25 microM) and 28B4 Fab (K(d) = 37 nM) to hapten 3. Nine replacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to its germline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 were characterized in detail in the contexts of both the germline and the affinity-matured antibodies. One of the mutations, Asp95(H)Trp, leads to a change in the orientation of the bound hapten, and its presence is a prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody for hapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwise manner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germline antibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities were found to be highly homologous to the germline antibody of 28B4, consistent with the notion that certain germline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg, F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. (1998) Science 279, 1929]. The ultimate specificity of the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somatic mutation. Insights into the evolution of antibody-combining sites provided by this and other structural studies are discussed. 相似文献
9.
V P Zav'yalov V M Abramov V G Skvortzov G V Troitsky 《Biochimica et biophysica acta》1977,493(2):359-366
The conformational changes of antibody structure induced by hapten molecule binding were investigated by means of thermal perturbation difference spectroscopy. The studies of the free rabit anti-dinitrophenyl antibodies show the conformational transition at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of the significant part of exposed tyrosine residues. Binding of the hapten molecules induces a similar transition to that which occurs between the two temperature dependent states of the free antibody. In contrast to our previous results with anti-dansyl rabbit antibodies the dinitrophenyl lysine stabilizes the "low temperature" native state of the protein. The investigation of the MOPC-315 mouse immunoglobulin A myeloma protein possessing anti-dinitrophenyl activity indicates no conformational transition at temperatures between 25 and 35 degrees C and only a small decrease of tyrosine exposure induced by the hapten binding. Our present and previous results indicate that most of the free immunoglobulins exist in two native conformational states which have a small difference in free energy. Hapten binding causes the transition in equilibrium between the two states towards the one of better binding. It is possible that this transition is necessary but not sufficient step for inducing the effector function of antibodies. 相似文献
10.
S Gilmour J Randall J Torbet R A Dwek S Wain-Hobson S K Dower M R van Schravendijk 《Proceedings of the Royal Society of London. Series B, Containing papers of a Biological character. Royal Society (Great Britain)》1981,211(1185):433-453
The first small-angle scattering study of an immunoglobulin A is reported. Neutron measurements have been made to determine conformational parameters of the mouse myeloma protein MOPC315 and to relate these to previous immunoglobulin G results. Use of the contrast method shows that the MOPC315 IgA molecule is not simply globular, that it has a dry volume of 220.0 +/- 4.5 nm3 corresponding to a mass density of 1.275 +/- 0.025 g cm-3 and that its full and cross-sectional radii of gyration, corrected for concentration dependence, are 7.97 +/- 0.07 nm, 2.40 +/- 0.08 nm and 1.33 +/- 0.07 nm respectively. Similar study of its Fab fragment gives a dry molecular volume of 69.0 +/- 0.7 nm3, a mass density of 1.285 +/- 0.015 g cm-3 and uncorrected radii of gyration that are consistent with those of the parent and support an overall "T" or "Y" conformation in solution. Addition to saturation of a small monovalent dinitrophenyl hapten leaves the dry volume of the whole molecule unaltered, but may slightly lower one or more of its radii of gyration. The significance of this finding is discussed. Comparative studies with rabbit anti-dinitrophenyl immunoglobulin G antibody suggest a different initial conformation but similar consequences of hapten binding, which, if real, are probably unrelated to classical complement fixation. 相似文献
11.
The magnetic field dependence of the proton-spin-lattice relaxation rate in rotationally immobilized proteins shows that the one-dimensional character of the protein primary structure causes a dramatic increase in the population of low-frequency motions from 10 kHz to 20 MHz. As a consequence, the probability and rate at which functionally critical conformational states are thermally sampled in a protein are dramatically increased as well, when compared with a three-dimensional lattice structure. Studies of protein dynamics often focus on time periods far shorter than those associated with catalytic function, but we show here that the magnetic field dependence of the proton nuclear spin-lattice relaxation rate in rotationally immobilized proteins reports unambiguously the structural fluctuations in the frequency range from 10 kHz to 20 MHz. This relaxation rate decreases with increasing Larmor frequency according to a power law that derives from the distribution of dynamical states, the localization of the structural disturbances, and the spatial distribution of hydrogen atoms in the structure. The robust theoretical foundation for the spin-relaxation process, loosely characterized as a direct spin-phonon coupling, shows that the disturbances propagate in a space of reduced dimensionality, essentially along the stiff connections of the polypeptide chain. The reduced dimensionality traps the disturbance and changes the efficiency for energy redistribution in the protein and the processes that drive nuclear spin relaxation. We also show that the Larmor frequency dependence of the protein-proton-spin-lattice relaxation rate constant is related to the frequency dependence of force constants and mean-square displacement commonly observed or calculated for proteins. We believe that these approaches give additional physical insight into the character of the extremely low-frequency protein dynamics. 相似文献
12.
A key problem in experimental protein folding is that of characterizing the conformational ensemble of denatured proteins under folding conditions. We address this problem by studying the conformational propensities of reductively unfolded RNase A under folding conditions, since earlier work has indicated that the equilibrium conformational ensemble of fully reduced RNase A resembles the transient conformational ensemble of a burst-phase folding intermediate of disulfide-intact RNase A. To assess these propensities, the relative disulfide-bond populations of the 1S, 2S, and 3S ensembles of the [C40A,C95A] mutant of RNase A were measured. Thirteen of the fifteen possible disulfide bonds are observed, consistent with earlier results and with the rapid reshuffling and lack of stable tertiary structure in these ensembles. This broad distribution contradicts recent observations by another group, but rigorous cross-checks show unambiguously that our data are self-consistent whereas their data are not. The distributions of disulfide bonds in the wild-type and mutant proteins show a power-law dependence on loop length, with an exponent that is significantly smaller than the exponents of either ideal or excluded-volume polymers. The 65-72 disulfide bond is much more strongly favored than would be predicted by this power law, consistent with earlier peptide studies and the disulfide-bond distributions of the 1S and 2S ensembles in wild-type RNase A. Experimental evidence suggests that this preference results from conformational biases in the backbone, rather than from differing accessibilities or reactivities of the two cysteine residues. In general, the other disulfide species do not deviate significantly from the power-law dependence, indicating that the conformational biases are relatively weak. 相似文献
13.
T P Theriault D J Leahy M Levitt H M McConnell G S Rule 《Journal of molecular biology》1991,221(1):257-270
Nuclear magnetic resonance has been used to study the structure of the anti-spin label antibody AN02 combining site and kinetic rates for the hapten-antibody reaction. The association reaction for the hapten dinitrophenyl-diglycine (DNP-diGly) is diffusion-limited. The activation enthalpy for association, 5.1 kcal/mol, is close to the activation enthalpy for diffusion in water. Several reliable resonance assignments have been made with the aid of recently reported crystal structure. Structural data deduced from the nuclear magnetic resonance (n.m.r.) spectra compare favorably with the crystal structure in terms of the combining site amino acid composition, distances of tyrosine residues from the unpaired electron of the hapten, and residues in direct contact with the hapten. Evidence is presented that a single binding site region tyrosine residue can assume two distinct conformations on binding of DNP-diGly. The AN02 antibody is an autoantibody. Dimerization of the Fab fragments is blocked by the hapten DNP-diGly. The n.m.r. spectra suggests that some of the amino acid residues involved in the binding of the DNP-hapten are also involved in the Fab dimerization. 相似文献
14.
Sabo TM Bakhtiari D Walter KF McFeeters RL Giller K Becker S Griesinger C Lee D 《Protein science : a publication of the Protein Society》2012,21(4):562-570
Physiological processes such as protein folding and molecular recognition are intricately linked to their dynamic signature, which is reflected in their thermal coefficient. In addition, the local conformational entropy is directly related to the degrees of freedom, which each residue possesses within its conformational space. Therefore, the temperature dependence of the local conformational entropy may provide insight into understanding how local dynamics may affect the stability of proteins. Here, we analyze the temperature dependence of internal methyl group dynamics derived from the cross-correlated relaxation between dipolar couplings of two CH bonds within ubiquitin. Spanning a temperature range from 275 to 308 K, internal methyl group dynamics tend to increase with increasing temperature, which translates to a general increase in local conformational entropy. With this data measured over multiple temperatures, the thermal coefficient of the methyl group order parameter, the characteristic thermal coefficient, and the local heat capacity were obtained. By analyzing the distribution of methyl group thermal coefficients within ubiquitin, we found that the N-terminal region has relatively high thermostability. These results indicate that methyl groups contribute quite appreciably to the total heat capacity of ubiquitin through the regulation of local conformational entropy. 相似文献
15.
We studied a pair of homologous thermophilic and mesophilic ribonuclease HI enzymes by molecular dynamics simulations. Each protein was subjected to three 5 ns simulations in explicit water at both 310 K and 340 K. The thermophilic enzyme showed larger overall positional fluctuations at both temperatures, while only the mesophilic enzyme at the higher temperature showed significant instability. When the temperature is changed, the relative flexibility of different local segments on the two proteins changed differently. Principal component analysis showed that the simulations of the two proteins explored largely overlapping regions in the conformational space. However, at 340 K, the collective structure variations of the thermophilic protein are different from those of the mesophilic protein. Our results, although not in accordance with the view that hyperthermostability of proteins may originate from their conformational rigidity, are consistent with several recent experimental and simulation studies which showed that thermophilic proteins may be conformationally more flexible than their mesophilic counterparts. The decorrelation between conformational rigidity and hyperthermostability may be attributed to the temperature dependence and long range nature of electrostatic interactions that play more important roles in the structural stability of thermophilic proteins. 相似文献
16.
Tong Li Malgorzata B. Tracka Shahid Uddin Jose Casas-Finet Donald J. Jacobs Dennis R. Livesay 《PLoS computational biology》2015,11(7)
The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation. 相似文献
17.
G protein alpha subunits mediate activation of signaling pathways through G protein-coupled receptors (GPCR) by virtue of GTP-dependent conformational rearrangements. It is known that regions of disorder in crystal structures can be indicative of conformational flexibility within a molecule, and there are several such regions in G protein alpha subunits. The amino-terminal 29 residues of Galpha are alpha-helical only in the heterotrimer, where they contact the side of Gbeta, but little is known about the conformation of this region in the active GTP bound state. To address the role of the Galpha amino-terminus in G-protein activation and to investigate whether this region undergoes activation-dependent conformational changes, a site-directed cysteine mutagenesis study was carried out. Engineered Galpha(i1) proteins were created by first removing six native reactive cysteines to yield a mutant Galpha(i1)-C3S-C66A-C214S-C305S-C325A-C351I that no longer reacts with cysteine-directed labels. Several cysteine substitutions along the amino-terminal region were then introduced. All mutant proteins were shown to be folded properly and functional. An environmentally sensitive probe, Lucifer yellow, linked to these sites showed a fluorescence change upon interaction with Gbetagamma and with activation by AlF(4)(-). Other fluorescent probes of varying charge, size, and hydrophobicity linked to amino-terminal residues also revealed changes upon activation with bulkier probes reporting larger changes. Site-directed spin-labeling studies showed that the N-terminus of the Galpha subunit is dynamically disordered in the GDP bound state, but adopts a structure consistent with an alpha-helix upon interaction with Gbetagamma. Interaction of the resulting spin-labeled Galphabetagamma with photoactivated rhodopsin, followed by rhodopsin-catalyzed GTPgammaS binding, caused the amino-terminal domain of Galpha to revert to a dynamically disordered state similar to that of the GDP-bound form. Together these results suggest conformational changes occur in the amino-termini of Galpha(i) proteins upon subunit dissociation and upon activating conformational changes. These solution studies reveal insights into conformational changes that occur dynamically in solution. 相似文献
18.
Conformational changes in human integrin alphaIIbbeta3 after platelet activation, monitored by FRET 总被引:1,自引:0,他引:1
Integrin alpha(IIb)beta(3), an abundant heterodimeric receptor at the surface of blood platelets, binds adhesive proteins after platelet activation and plays a primary role in haemostasis. In solution, it has been observed mainly in two conformations: the bent and the extended forms. Based on X-ray crystallography, electron microscopy and immunochemical observations of full-length integrin ectodomains and intact integrins, it has been agreed that unactivated integrins are in the bent conformation, both isolated in solution and in living cells. However, consensus is yet to emerge on the bent or extended conformation of activated integrins and on their mechanism of activation (the switchblade, the deadbolt and the S-S reduction models), which require further experimental tests at the cell level to become established facts. Here, we tested the proposed structural rearrangements undergone by integrin alpha(IIb)beta(3) after cell activation, by using F?rster-type fluorescence resonance energy transfer (FRET) and attached fluorescent labels to Fab fragments of monoclonal antibodies directed to the betaA domain of the beta(3) subunit (donor, Alexa488-P97 Fab) and to the Calf-2 domain of the alpha(IIb) subunit (acceptor, Cy3-M3 Fab or Cy3-M10 Fab). The FRET efficiencies observed after ADP or TRAP platelet activation changed less than 20% from the resting values, showing that the distance between the labeled Fab fragments changes only modestly after platelet activation by physiological agonists. This observation is consistent with a conformational model of the activated integrin in the cell less extended than in the switchblade model. 相似文献
19.
N M Kirillova O V Zhorov V A Pre?gerzon S P Martsev 《Biokhimii?a (Moscow, Russia)》1991,56(6):1057-1068
The affinity-purified by chromatography on immobilized antigen rabbit IgG was modified with mixed carboxycarbonic anhydride of DTPA which markedly alters the interaction of charged residues in the protein molecule. To study the correlation between the antigen binding activity and the conformational mobility of IgG, the reactivity of modified IgG towards conformational probes targeted at variable and constant IgG domains, was investigated. The antibody against CH2 domains of IgG, staphylococcal protein A and protein antigen ferritin were used as conformational probes. It was found that modification of IgG amino groups entails the global increase in conformational mobility involving the Fab fragments, CH2 and, probably, the CH3 domains of the Fc portion of IgG. Taking advantage of Fab fragments modification it was shown that two processes contribute to the global increase in the conformational mobility of IgG. These processes are: i) stimulation of segmental flexibility and, ii) increase in the mobility within the Fv domains of the Fab fragments. 相似文献
20.
We introduce a simple procedure to analyze the temperature dependence of the folding and unfolding rates of two-state proteins. We start from the simple transition-state-like rate expression: k = D(eff)exp(-DeltaG(TS)/RT), in which upper and lower bounds for the intra-chain effective diffusion coefficient (D(eff)) are obtained empirically using the timescales of elementary processes in protein folding. From the changes in DeltaG(TS) as a function of temperature, we calculate enthalpies and heat capacities of activation, together with the more elusive entropies of activation. We then estimate the conformational entropy of the transition state by extrapolation to the temperature at which the solvation entropy vanishes by cancellation between polar and apolar terms. This approach is based on the convergence temperatures for the entropy of solvating apolar (approximately 385 K) and polar groups (approximately 335 K), the assumption that the structural properties of the transition state are somewhere in between the unfolded and folded states, and the established relationship between observed heat capacity and solvent accessibility.1 To circumvent the lack of structural information about transition states, we use the empirically determined heat capacities of activation as constraints to identify the extreme values of the transition state conformational entropy that are consistent with experiment. The application of this simple approach to six two-state folding proteins for which there is temperature-dependent data available in the literature provides important clues about protein folding. For these six proteins, we obtain an average equilibrium cost in conformational entropy of -4.3 cal x mol(-1)K(-1)per residue, which is in close agreement to previous empirical and computational estimates of the same quantity. Furthermore, we find that all these proteins have a conformationally diverse transition state, with more than half of the conformational entropy of the unfolded state. In agreement with predictions from theory and computer simulations, the transition state signals the change from a regime dominated by loss in conformational entropy to one driven by the gain in stabilization free energy (i.e., including protein interactions and solvation effects). Moreover, the height of the barrier is determined by how much stabilization free energy is realized at that point, which is related to the relative contribution of local versus non-local interactions. A remarkable observation is that the fraction of conformational entropy per residue that is present in the transition state is very similar for the six proteins in this study. Based on this commonality, we propose that the observed change in thermodynamic regime is connected to a change in the pattern of structure formation: from one driven by formation of pairwise interactions to one dominated by coupling of the networks of interactions involved in forming the protein core. In this framework, the barrier to two-state folding is crossed when the folding protein reaches a "critical native density" that allows expulsion of remaining interstitial water and consolidation of the core. The principle of critical native density should be general for all two-state proteins, but can accommodate different folding mechanisms depending on the particularities of the structure and sequence. 相似文献