RESUMO
(15)N relaxation dispersion experiments were applied to the isolated N-terminal SH3 domain of the Drosophila protein drk (drkN SH3) to study microsecond to second time scale exchange processes. The drkN SH3 domain exists in equilibrium between folded (F(exch)) and unfolded (U(exch)) states under nondenaturing conditions in a ratio of 2:1 at 20 degrees C, with an average exchange rate constant, k(ex), of 2.2 s(-1) (slow exchange on the NMR chemical shift time scale). Consequently a discrete set of resonances is observed for each state in NMR spectra. Within the U(exch) ensemble there is a contiguous stretch of residues undergoing conformational exchange on a micros/ms time scale, likely due to local, non-native hydrophobic collapse. For these residues both the F(exch) <--> U(exch) conformational exchange process and the micros/ms exchange event within the U(exch) state contribute to the (15)N line width and can be analyzed using CPMG-based (15)N relaxation dispersion measurements. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental (15)N relaxation dispersion data with results from an (15)N longitudinal relaxation experiment that accurately quantifies exchange rates in slow exchanging systems (Farrow, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol. NMR 1994, 4, 727-734). A simple, generally applicable analytical expression for the dependence of the effective transverse relaxation rate constant on the pulse spacing in CPMG experiments has been derived for a two-state exchange process in the slow exchange limit, which can be used to fit the experimental data on the global folding/unfolding transition. The results illustrate that relaxation dispersion experiments provide an extremely sensitive tool to probe conformational exchange processes in unfolded states and to obtain information on the free energy landscape of such systems.
Assuntos
Proteínas de Drosophila , Proteínas de Insetos/química , Domínios de Homologia de src , Animais , Drosophila , Cinética , Computação Matemática , Modelos Químicos , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
Protein structure is inherently dynamic, with function often predicated on excursions from low to higher energy conformations. For example, X-ray studies of a cavity mutant of T4 lysozyme, L99A, show that the cavity is sterically inaccessible to ligand, yet the protein is able to bind substituted benzenes rapidly. We have used novel relaxation dispersion NMR techniques to kinetically and thermodynamically characterize a transition between a highly populated (97%, 25 degrees C) ground state conformation and an excited state that is 2.0 kcal mol(-1) higher in free energy. A temperature-dependent study of the rates of interconversion between ground and excited states allows the separation of the free energy change into enthalpic (Delta H = 7.1 kcal mol(-1)) and entropic (T Delta S = 5.1 kcal mol(-1), 25 degrees C) components. The residues involved cluster about the cavity, providing evidence that the excited state facilitates ligand entry.
Assuntos
Bacteriófago T4/enzimologia , Espectroscopia de Ressonância Magnética , Muramidase/química , Muramidase/metabolismo , Substituição de Aminoácidos/genética , Bacteriófago T4/genética , Sítios de Ligação , Entropia , Cinética , Ligantes , Modelos Moleculares , Movimento (Física) , Muramidase/genética , Estrutura Secundária de Proteína , TemperaturaRESUMO
p53 is a nuclear phosphoprotein that regulates cellular fate after genotoxic stress through its role as a transcriptional regulator of genes involved in cell cycle control and apoptosis. The C-terminal region of p53 is known to negatively regulate sequence specific DNA-binding of p53; modifications to the C-terminus relieve this inhibition. Two models have been proposed to explain this latency: (i) an allosteric model in which the C-terminal domain interacts with another domain of p53 or (ii) a competitive model in which the C-terminal and the core domains compete for DNA binding. We have characterized latent and active forms of dimeric p53 using gel mobility shift assays and NMR spectroscopy. We show on the basis of chemical shifts that dimeric p53 both containing and lacking the C-terminal domain are identical in conformation and that the C-terminus does not interact with other p53 domains. Similarly, NMR spectra of isolated core and tetramerization domains confirm a modular p53 architecture. The data presented here rule out an allosteric model for the regulation of p53.
Assuntos
Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Regulação Alostérica , Sítios de Ligação , DNA/química , DNA/genética , DNA/metabolismo , Dimerização , Humanos , Modelos Biológicos , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Deleção de Sequência/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
A new NMR experiment is presented for the measurement of micros-ms time scale dynamics of Asn and Gln side chains in proteins. Exchange contributions to the (15)N line widths of side chain residues are determined via a relaxation dispersion experiment in which the effective nitrogen transverse relaxation rate is measured as a function of the number of refocusing pulses in constant-time, variable spacing CPMG intervals. The evolution of magnetization from scalar couplings and dipole-dipole cross-correlations, which has limited studies of exchange in multi-spin systems in the past, does not affect the extraction of accurate exchange parameters from relaxation profiles of NH(2) groups obtained in the present experiment. The utility of the method is demonstrated with an application to a Leu --> Ala cavity mutant of T4 lysozyme, L99A. It is shown that many of the side chain amide groups of Asn and Gln residues in the C-terminal domain of the protein are affected by a chemical exchange process which may be important in facilitating the rapid binding of hydrophobic ligands to the cavity.
Assuntos
Asparagina/química , Bacteriófago T4/enzimologia , Glicina/química , Muramidase/química , Mutação , Proteínas/química , Modelos Moleculares , Muramidase/genética , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Difração de Raios XRESUMO
A relaxation dispersion-based NMR experiment is presented for the measurement and quantitation of micros-ms dynamic processes at methyl side-chain positions in proteins. The experiment measures the exchange contribution to the 13C line widths of methyl groups using a constant-time CPMG scheme. The effects of cross-correlated spin relaxation between dipole-dipole and dipole-CSA interactions as well as the effects of scalar coupling responsible for mixing of magnetization modes during the course of the experiment have been investigated in detail both theoretically and through simulations. It is shown that the complex relaxation properties of the methyl spin system do not complicate extraction of accurate exchange parameters as long as care is taken to ensure that appropriate magnetization modes are interchanged in the middle of the constant-time CPMG period. An application involving the measurement of relaxation dispersion profiles of methionine residues in a Leu99Ala substitution of T4 lysozyme is presented. All of the methionine residues are sensitive to an exchange event with a rate on the order of 1200 s(-1) at 20 degrees C that may be linked to a process in which hydrophobic ligands are able to rapidly bind to the cavity that is present in this mutant.
Assuntos
Metionina/química , Muramidase/química , Algoritmos , Ligantes , Espectroscopia de Ressonância Magnética , Muramidase/genética , MutaçãoRESUMO
The p53 tumor suppressor is a transcription factor that plays a crucial role in the activation of genes in response to DNA damage. As a first step towards detailed structural studies of the molecule aimed at understanding its regulation, we have used 4D-TROSY triple resonance NMR spectroscopy to obtain nearly complete 1H(N), 15N, 13C(alpha), 13CO and 13C(beta) resonance assignments of a dimeric form of the protein comprising DNA-binding and oligomerization domains (67 kDa). A simple comparison of 4D spectra recorded on p53 molecules consisting of DNA-binding and oligomerization domains with and without the regulatory domain establishes that both constructs have essentially identical chemical shifts. Although the affinity of p53 for target DNA is decreased in constructs containing the regulatory domain, the chemical shift results reported here suggest that this decrease is not due to specific domain interactions involving the regulatory portion of the molecule, or alternatively, that such interactions require the presence of DNA.
Assuntos
Proteína Supressora de Tumor p53/química , Sítios de Ligação , Isótopos de Carbono , Monóxido de Carbono , DNA/química , DNA/metabolismo , Dimerização , Hidrogênio , Substâncias Macromoleculares , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Proteína Supressora de Tumor p53/metabolismoRESUMO
The Leu99-->Ala mutant of T4 lysozyme contains a large internal cavity in the core of its C-terminal domain that is capable of reversibly binding small hydrophobic compounds. Although the cavity is completely buried, molecules such as benzene or xenon can exchange rapidly in and out. The dynamics of the unliganded protein have been compared to the wild-type protein by measuring the NMR spin relaxation rates of backbone amide and side chain methyl nuclei. Many residues surrounding the cavity were found to be affected by a chemical exchange process with a rate of 1500 +/- 200 s(-1), which is quenched upon addition of saturating amounts of the ligand xenon. The relationship between the structure, dynamics, and energetics of the T4 lysozyme mutant is discussed.
Assuntos
Bacteriófago T4/enzimologia , Bacteriófago T4/genética , Muramidase/química , Muramidase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alanina/genética , Substituição de Aminoácidos/genética , Isótopos de Carbono , Cristalografia por Raios X , Hidrogênio , Leucina/genética , Ligantes , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica/genética , Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Prótons , Termodinâmica , Xenônio/químicaRESUMO
The retinoid X receptor (RXR) is a prominent member of the nuclear receptor family of ligand-inducible transcription factors. Many proteins of this family exert their function as heterodimers with RXR as a common upstream partner. Studies of the DNA-binding domains of several nuclear receptors reveal differences in structure and dynamics, both between the different proteins and between the free- and DNA-bound receptor DBDs. We investigated the differences in dynamics between RXR free in solution and in complex with a 14 base-pair oligonucleotide, using (1)H and (15)N relaxation studies. Nano- to picosecond dynamics were probed on (15)N, employing Lipari-Szabo analysis with an axially symmetric tumbling model to estimate the exchange contributions to the transverse relaxation rates. Furthermore, milli- to microsecond dynamics were estimated qualitatively for (1)H and (15)N, using CPMG-HSQC and CPMG-T(2) measurements with differential pulse spacing. RXR shows hardly any nano- to picosecond time-scale internal motion. Upon DNA binding, the order parameters show a tiny increase. Dynamics in the milli- to microsecond time scale is more prevalent. It is localized in the first and second zinc fingers of the free RXR. Upon DNA-binding, exchange associated with specific/aspecific DNA-binding of RXR is observed throughout the sequence, whereas conformational flexibility of the D-box and the second zinc finger of RXR is greatly reduced. Since this DNA-binding induced folding transition occurs remote from the DNA in a region which is involved in protein-protein interactions, it may very well be related to the cooperativity of dimeric DNA binding.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Oligodesoxirribonucleotídeos/química , Receptores do Ácido Retinoico/química , Fatores de Transcrição/química , Anisotropia , Pareamento de Bases , Sítios de Ligação , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Difusão , Dimerização , Cinética , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/metabolismo , Estrutura Terciária de Proteína , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Soluções , Termodinâmica , Fatores de Transcrição/metabolismoRESUMO
High-alkaline serine proteases have been successfully applied as protein degrading components of detergent formulations and are subject to extensive protein engineering efforts to improve their stability and performance. Dynamics has been suggested to play an important role in determining enzyme activity and specificity and it is therefore of interest to establish how local changes in internal mobility affect protein stability, specificity and performance. Here we present the dynamic properties of the 269 residue serine proteases subtilisin PB92 (Maxacal(TM)) and subtilisin BLS (Savinase(TM)), secreted by Bacillus lentus, and an engineered quadruple variant, DSAI, that has improved washing performance. T1, T2 and heteronuclear NOE measurements of the 15N nuclei indicate that for all three proteins the majority of the backbone is very rigid, with only a limited number of residues being involved in local mobility. Many of the residues that constitute the S1 and S4 pockets, determining substrate specificity, are flexible in solution. In contrast, the backbone amides of the residues that constitute the catalytic triad do not exhibit any motion. Subtilisins PB92, BLS and DSAI demonstrate similar but not identical NMR relaxation rates. A detailed analysis of local flexibility indicates that the motion of residues Thr143 and Ala194 becomes more restricted in subtilisin BLS and DSAI. Noteworthy, the loop regions involved in substrate binding become more structured in the engineered variant as compared with the two native proteases, suggesting a relation between altered dynamics and performance. Similar conclusions have been established by X-ray crystallograpic methods, as shown in the accompanying paper.
Assuntos
Bacillus/enzimologia , Serina Endopeptidases/química , Subtilisinas/química , Sítios de Ligação , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Engenharia de Proteínas , Estrutura Terciária de Proteína , Prótons , Serina Endopeptidases/genética , Especificidade por Substrato , Subtilisinas/genéticaRESUMO
Slow protein dynamics can be studied by 15N spin-echo (CPMG) and off-resonance rotating frame relaxation through the effective field dependence of the exchange-mediated relaxation contribution. It is shown that, by a combination of these complementary techniques, a more extended sampling of the microsecond time scale processes is achieved than by either method alone. 15N R2 and improved off-resonance R1 rho experiments [Mulder et al. (1998) J. Magn. Reson., 131, 351-357] were applied to the 9-cis-retinoic acid receptor DNA-binding domain and allowed the identification of 14 residues exhibiting microsecond time scale dynamics. Assuming exchange between two conformational substates, average lifetimes ranging from 37 to 416 microseconds, and chemical shift differences of up to 3 ppm were obtained. The largest perturbation of tertiary structure was observed for the second zinc finger region, which was found to be disordered in the solution structure [Holmbeck et al. (1998) J. Mol. Biol., 281, 271-284]. Since this zinc-coordinating domain comprises the principal dimerization interface for RXR in a wide repertoire of complexes with different hormone receptors to their cognate response elements, this finding has important implications for our understanding of nuclear receptor assembly on DNA direct repeats. The flexibility observed for the dimerization domain may explain how RXR, through the ability to adaptively interact with a wide variety of highly homologous partner molecules, demonstrates such a versatile DNA-binding repertoire.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Receptores do Ácido Retinoico/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Sítios de Ligação , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dimerização , Hidrogênio , Cinética , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Estrutura Terciária de Proteína , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Rotação , Soluções , Fatores de Tempo , Fatores de Transcrição/metabolismo , Zinco/metabolismoRESUMO
The all-trans retinoic acid and 9-cis retinoic acid receptors (RAR and RXR, respectively) belong to a family of ligand inducible transcription factors, which exert their effect via binding to hormone response elements. Both are members of the class II sub-family of nuclear receptors, which bind DNA as dimers, on tandem repeats of a hexamer motif separated by a variable spacer. The variability in spacer length and the head-to-tail organization of the hormone response elements result in different protein-protein interactions in each of the complexes. We show that the zinc-coordinating loop regions of RXR and RAR DNA-binding domains exhibit dynamics on the millisecond to microsecond time scale. The highly dynamic second zinc finger of RXR constitutes the primary protein-protein interface in many nuclear receptor assemblies on DNA. Dynamics is also observed in the first and second zinc fingers of RAR, which are implicated in dimeric interactions with RXR on response elements with spacers of 5 base pairs and 1 base pair, respectively. The striking correspondence between the regions that exhibit conformational exchange and the dimer interfaces of the proteins complexed with DNA suggests a functional role for the dynamics. The observed flexibility may allow the proteins to adapt to various partners and with different orientations upon assembly on DNA. Furthermore, the more extensive dynamics observed for RXR may reflect the greater ability of this protein to modulate its interaction surface since it participates in a wide variety of receptor complexes.
Assuntos
Proteínas de Ligação a DNA/química , DNA/metabolismo , Receptores do Ácido Retinoico/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteínas de Ligação a DNA/metabolismo , Dimerização , Humanos , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Termodinâmica , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
The solution structure of photoactive yellow protein (PYP), a photosensory protein from Ectothiorhodospira halophila, has been determined by multidimensional NMR spectroscopy. The structure consists of an open, twisted, 6-stranded, antiparallel beta-sheet, which is flanked by four alpha-helices on both sides. The final set of 26 selected structures is well-defined for the regions spanning residues Phe6-Ala16, Asp24-Ala112, and Tyr118-Val125 and displays a root-mean-square deviation, versus the average, of 0.45 A for the backbone and 0.88 A for all heavy atoms. Comparison of the solution structure with an earlier published 1.4 A crystal structure (Borgstahl, G. E. O., Williams, D. R., and Getzoff, E. D. (1995) Biochemistry 34, 6278-6287) reveals a similarity with a root-mean-square deviation of 1.77 A for the backbone for the well-defined regions. The most distinct difference in the backbone with the crystal structure is found near the N-terminus, for residues Asp19-Leu23, which corresponds to an alpha-helix in the crystal structure and to one of the poorest defined regions in the solution structure. To characterize the dynamic behavior of PYP in solution, we undertook a 15N relaxation study and measurements of hydrogen/deuterium exchange. Determination of order parameters through the model-free Lipari-Szabo approach enabled the identification of several regions of enhanced dynamics. The comparison of atomic displacements in the backbone traces of the ensemble structures, with mobility measurements from NMR, show that the poorly defined regions feature fast internal motions in the nanosecond to picosecond time scale.
Assuntos
Proteínas de Bactérias/química , Fotorreceptores Microbianos , Conformação Proteica , Termodinâmica , Chromatiaceae/química , Cristalização , Cristalografia por Raios X , Deutério , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Prótons , SoluçõesRESUMO
Light irradiation of photoactive yellow protein (PYP) induces a photocycle, in which red-shifted (pR) and blue-shifted (pB) intermediates have been characterized. An NMR study of the long-lived pB intermediate now reveals that it exhibits a large degree of disorder and exists as a family of multiple conformers that exchange on a millisecond time scale. This shows that the behavior of PYP in solution is different from what has been observed in the crystalline state. Furthermore, differential refolding to ground state pG is observed, whereby the central beta-sheet and parts of the helical structure are formed first and the region around the chromophore at a later stage.
Assuntos
Proteínas de Bactérias/química , Fotorreceptores Microbianos , Conformação Proteica , Proteínas de Bactérias/efeitos da radiação , Chromatiaceae/química , Lasers , Ressonância Magnética Nuclear BiomolecularRESUMO
BACKGROUND: Research on high-alkaline proteases, such as serine protease PB92, has been largely inspired by their industrial application as protein-degrading components of washing powders. Serine protease PB92 is a member of the subtilase family of enzymes, which has been extensively studied. These studies have included exhaustive protein engineering investigations and X-ray crystallography, in order to provide insight into the mechanism and specificity of enzyme catalysis. Distortions have been observed in the substrate-binding region of subtilisin crystal structures, due to crystal contacts. In addition, the structural variability in the substrate-binding region of subtilisins is often attributed to flexibility. It was hoped that the solution structure of this enzyme would provide further details about the conformation of this key region and give new insights into the functional properties of these enzymes. RESULTS: The three-dimensional solution structure of the 269-residue (27 kDa) serine protease PB92 has been determined using distance and dihedral angle constraints derived from triple-resonance NMR data. The solution structure is represented by a family of 18 conformers which overlay onto the average structure with backbone and all-heavy-atom root mean square deviations (for the main body of the molecule) of 0.88 and 1.21 A, respectively. The family of structures contains a number of regions of relatively high conformational heterogeneity, including various segments that are involved in the formation of the substrate-binding site. The presence of flexibility within these segments has been established from NMR relaxation parameters and measurements of amide proton exchange rates. CONCLUSIONS: The solution structure of the serine protease PB92 presents a well defined global fold which is rigid with the exception of a restricted number of sites. Among the limited number of residues involved in significant internal mobility are those of two pockets, termed S1 and S4, within the substrate-binding site. The presence of flexibility within the binding site supports the proposed induced fit mechanism of substrate binding.
Assuntos
Bacillus/enzimologia , Dobramento de Proteína , Estrutura Secundária de Proteína , Subtilisinas/química , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Engenharia de Proteínas , Soluções , Subtilisinas/isolamento & purificaçãoRESUMO
We present here HSQC experiments with improved sensitivity for signals in the presence of exchange broadening. During periods of coherence transfer through scalar coupling the experiments employ CPMG-derived pulse trains to reduce loss of dephasing of spin coherence due to chemical exchange. (15)N-(1)H gradient CPMG-HSQC and SE-CPMG-HSQC experiments have been developed and applied to complexes of lac repressor headpiece with operator DNA. Improved sensitivity is demonstrated for many protein backbone and side-chain resonances in the complex, markedly for signals of protons located at the protein-DNA interface. In addition, a significant increase in intensity is observed for arginine guanidino groups undergoing conformational exchange.