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1.
Proc Natl Acad Sci U S A ; 112(29): 9022-7, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26130807

RESUMO

The energy landscapes of proteins are highly complex and can be influenced by changes in physical and chemical conditions under which the protein is studied. The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein two electrons are transferred to the heme group and the enzyme visits several conformational states. Using paramagnetic NMR spectroscopy with a lanthanoid tag, we show that the enzyme bound to its redox partner, putidaredoxin, is in a closed state at ambient temperature in solution. This result contrasts with recent crystal structures of the complex, which suggest that the enzyme opens up when bound to its partner. The closed state supports a model of catalysis in which the substrate is locked in the active site pocket and the enzyme acts as an insulator for the reactive intermediates of the reaction.


Assuntos
Cânfora 5-Mono-Oxigenase/química , Pseudomonas putida/enzimologia , Cânfora/química , Ferredoxinas , Modelos Moleculares , Isótopos de Nitrogênio , Oxirredução , Conformação Proteica , Espectroscopia de Prótons por Ressonância Magnética , Especificidade por Substrato
2.
J Biomol NMR ; 61(2): 123-36, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25563704

RESUMO

NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxation dispersion is the increase in the amplitude of dispersion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dispersion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements.


Assuntos
Adenilato Quinase/química , Ressonância Magnética Nuclear Biomolecular/métodos , Adenilato Quinase/análise , Elementos da Série dos Lantanídeos/química , Modelos Moleculares , Conformação Proteica
3.
Chembiochem ; 15(1): 80-6, 2014 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-24302683

RESUMO

Cytochrome P450cam (P450cam) is a heme-containing monooxygenase that catalyzes the hydroxylation of D-camphor to produce 5-exo-hydroxycamphor. The catalytic cycle of P450cam requires two electrons, both of which are donated by putidaredoxin (Pdx), a ferredoxin containing a [2 Fe-2 S] cluster. Atomic-resolution structures of the Pdx-P450cam complex have recently been solved by X-ray crystallography and paramagnetic NMR spectroscopy. The binding interface showed the potential electron transfer pathways and interactions between Pdx Asp38 and P450cam Arg112, as well as hydrophobic contacts between the Pdx Trp106 and P450cam residues. Several polar residues not previously recognized as relevant for binding were found in the interface. In this study, site-directed mutagenesis, kinetic measurements, and NMR studies were employed to probe the energetic importance and role of the polar residues in the Pdx-P450cam interaction. A double mutant cycle (DMC) analysis of kinetic data shows that favorable interactions exist between Pdx Tyr33 and P450cam Asp125, as well as between Pdx Ser42 and P450cam His352. The results show that alanine substitutions of these residues and several others do not influence the rates of electron transfer. It is concluded that these polar interactions contribute to partner recognition rather than to electronic coupling of the redox centers.


Assuntos
Cânfora 5-Mono-Oxigenase/metabolismo , Ferredoxinas/metabolismo , Sítios de Ligação , Cânfora 5-Mono-Oxigenase/química , Cânfora 5-Mono-Oxigenase/genética , Transporte de Elétrons , Ferredoxinas/química , Ferredoxinas/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
4.
Chemistry ; 20(21): 6256-8, 2014 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-24737492

RESUMO

Paramagnetic NMR probes provide valuable long-range structural information on proteins and protein complexes. A new, stable, two-armed lanthanoid probe is reported that can be attached to a protein site-specifically via chemically inert thioether linkages.


Assuntos
Quelantes/química , Elementos da Série dos Lantanídeos/química , Modelos Moleculares , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular
5.
J Biomol NMR ; 55(4): 379-89, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23526169

RESUMO

The use of paramagnetic NMR data for the refinement of structures of proteins and protein complexes is widespread. However, the power of paramagnetism for protein assignment has not yet been fully exploited. PARAssign is software that uses pseudocontact shift data derived from several paramagnetic centers attached to the protein to obtain amide and methyl assignments. The ability of PARAssign to perform assignment when the positions of the paramagnetic centers are known and unknown is demonstrated. PARAssign has been tested using synthetic data for methyl assignment of a 47 kDa protein, and using both synthetic and experimental data for amide assignment of a 14 kDa protein. The complex fitting space involved in such an assignment procedure necessitates that good starting conditions are found, both regarding placement and strength of paramagnetic centers. These starting conditions are obtained through automated tensor placement and user-defined tensor parameters. The results presented herein demonstrate that PARAssign is able to successfully perform resonance assignment in large systems with a high degree of reliability. This software provides a method for obtaining the assignments of large systems, which may previously have been unassignable, by using 2D NMR spectral data and a known protein structure.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Azurina/química , Sistema Enzimático do Citocromo P-450/química , Software
6.
J Am Chem Soc ; 134(41): 17306-13, 2012 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-22994925

RESUMO

Paramagnetic lanthanides ions are broadly used in NMR spectroscopy. The effects of unpaired electrons on NMR spectral parameters provide a powerful tool for the characterization of macromolecular structures and dynamics. Here, a new lanthanide-chelating NMR probe, Caged Lanthanide NMR Probe-7 (CLaNP-7), is presented. It can be attached to protein surfaces via two disulfide bridges, yielding a probe that is rigid relative to the protein backbone. CLaNP-7 extends the application range of available probes. It has a yellow color, which is helpful for sample preparation. Its effects are comparable to those of CLaNP-5, but its charge is two units lower (+1) than that of CLaNP-5 (+3), reducing the change in surface potential after probe attachment. It also has a different magnetic susceptibility tensor, so by using both tags, two sets of structural restraints can be obtained per engineered cysteine pair. Moreover, it was found that the orientation of the magnetic susceptibility tensor is pH dependent (pK(a) ≈ 7) when a histidine residue is located in the neighborhood of the probe attachment site. The results show that the His imidazole group interacts with the CLaNP-7 tag. It is proposed that the histidine residue forms a hydrogen bond to a water/hydroxyl molecule that occupies the ninth coordination position on the lanthanide, thus breaking the two-fold symmetry of the CLaNP tag in a pH-dependent way.


Assuntos
Quelantes/química , Corantes Fluorescentes/química , Elementos da Série dos Lantanídeos/química , Ressonância Magnética Nuclear Biomolecular , Quelantes/síntese química , Corantes Fluorescentes/síntese química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Proteínas/química
7.
J Am Chem Soc ; 132(29): 9952-3, 2010 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-20586489

RESUMO

Paramagnetic lanthanide tags potentially can enhance the effects of microsecond to millisecond dynamics in proteins on NMR signals and provide structural information on lowly populated states encoded in the pseudocontact shifts. We have investigated the microsecond to millisecond mobility of a two-point attached lanthanide tag, CLaNP-5, using paramagnetic (1)H CPMG relaxation dispersion methods. CLaNP-5 loaded with Lu(3+), Yb(3+), or Tm(3+) was attached to three sites on the surface of two proteins, pseudoazurin and cytochrome c. The paramagnetic center causes large relaxation dispersion effects for two attachment sites, suggesting that local dynamics of the protein at the attachment site causes mobility of the paramagnetic center. At one site the relaxation dispersions are small and limited to the immediate environment of the tag. It is concluded that paramagnetic relaxation dispersion could represent a sensitive method to probe protein dynamics. However, the selection of a rigid attachment site is of critical importance.


Assuntos
Elementos da Série dos Lantanídeos/metabolismo , Magnetismo , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/metabolismo , Alcaligenes faecalis , Azurina/química , Azurina/metabolismo , Citocromos c/química , Citocromos c/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas/química , Reprodutibilidade dos Testes , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Biochemistry ; 48(1): 50-8, 2009 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-19072172

RESUMO

The dynamics of the reduced form of the blue copper protein pseudoazurin from Alcaligenes faecalis S-6 was investigated using (15)N relaxation measurements with a focus on the dynamics of the micro- to millisecond time scale. Different types of conformational exchange processes are observed in the protein on this time scale. At low pH, the protonation of the C-terminal copper-ligated histidine, His81, is observed. A comparison of the exchange rates in the presence and absence of added buffers shows that the protonation is the rate-limiting step at low buffer concentrations. This finding agrees with previous observations for other blue copper proteins, e.g., amicyanin and plastocyanin. However, in contrast to plastocyanin but similar to amicyanin, a second conformational exchange between different conformations of the protonated copper site is observed at low pH, most likely triggered by the protonation of His81. This process has been further characterized using CPMG dispersion methods and is found to occur with a rate of a few thousands per second. Finally, micro- to millisecond motions are observed in one of the loop regions and in the alpha-helical regions. These motions are unaffected by pH and are unrelated to the conformational changes in the active site of pseudoazurin.


Assuntos
Alcaligenes faecalis/química , Azurina/química , Cobre , Metaloproteínas/química , Soluções Tampão , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Conformação Proteica
9.
J Am Chem Soc ; 131(48): 17576-82, 2009 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-19908864

RESUMO

In oxygenic photosynthetic cells, carbon metabolism is regulated by a light-dependent redox signaling pathway through which the light signal is transmitted in the form of electrons via a redox chain comprising ferredoxin (Fd), ferredoxin:thioredoxin reductase (FTR), and thioredoxin (Trx). Trx affects the activity of a variety of enzymes via dithiol oxidation and reduction reactions. FTR reduces an intramolecular disulfide bridge of Trx, and Trx reduction involves a transient cross-link with FTR. NMR spectroscopy was used to investigate the interaction of Fd, FTR, and an m-type Trx. NMR titration experiments indicate that FTR uses distinct sites to bind Fd and Trx simultaneously to form a noncovalent ternary complex. The orientation of Trx-m relative to FTR was determined from the intermolecular paramagnetic broadening caused by the [4Fe-4S] cluster of FTR. Two models of the noncovalent binary complex of FTR/Trx-m based on the paramagnetic distance restraints were obtained. The models suggest that either a modest or major rotational movement of Trx must take place when the noncovalent binary complex proceeds to the covalent complex. This study demonstrates the complementarity of paramagnetic NMR and X-ray diffraction of crystals in the elucidation of dynamics in a transient protein complex.


Assuntos
Ferredoxinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Oxirredutases/metabolismo , Tiorredoxinas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Ferredoxinas/química , Proteínas Ferro-Enxofre/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Movimento , Oxirredutases/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ligação Proteica , Soluções , Spinacia oleracea , Synechocystis/enzimologia , Tiorredoxinas/química
10.
J Biomol NMR ; 44(4): 225-33, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19533375

RESUMO

The use of 13C NMR relaxation dispersion experiments to monitor micro-millisecond fluctuations in the protonation states of histidine residues in proteins is investigated. To illustrate the approach, measurements on three specifically 13C labeled histidine residues in plastocyanin (PCu) from Anabaena variabilis (A.v.) are presented. Significant Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion is observed for 13C(epsilon1) nuclei in the histidine imidazole rings of A.v. PCu. The chemical shift changes obtained from the CPMG dispersion data are in good agreement with those obtained from the chemical shift titration experiments, and the CPMG derived exchange rates agree with those obtained previously from 15N backbone relaxation measurements. Compared to measurements of backbone nuclei, 13C(epsilon1) dispersion provides a more direct method to monitor interchanging protonation states or other kinds of conformational changes of histidine side chains or their environment. Advantages and shortcomings of using the 13C(epsilon1) dispersion experiments in combination with chemical shift titration experiments to obtain information on exchange dynamics of the histidine side chains are discussed.


Assuntos
Histidina/química , Ressonância Magnética Nuclear Biomolecular/métodos , Plastocianina/química , Anabaena variabilis/química , Isótopos de Carbono/química , Concentração de Íons de Hidrogênio , Imidazóis/química , Modelos Químicos , Modelos Moleculares , Prótons , Temperatura
11.
Biochemistry ; 47(50): 13308-17, 2008 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-19086273

RESUMO

Analogous to insulin, the relaxin-like factor (RLF) must undergo a structural transition to the active form prior to receptor binding. Thus, the C-terminus of the B chain of RLF folds toward the surface of the central B chain helix, causing partial obliteration of the two essential RLF receptor-binding site residues, valine B19 and tryptophan B27. Via comparison of the solution structure of a fully active C-terminally cross-linked RLF analogue with the native synthetic human RLF (hRLF), it became clear that the cross-linked analogue largely retains the essential folding of the native protein. Both proteins exist in a major and minor conformation, as revealed by multiple resonances from tryptophan B27 and adjacent residues on the B chain helix. Notably, the minor conformation is significantly more highly populated in the chemically cross-linked RLF than it is in the hRLF. In addition, compared to the unmodified molecule, subtle differences are observed within the B chain helix whereby the cross-linked derivative shows a reduced level of hydrogen bonding and significant peak broadening at the binding site residue ValB19. On the basis of these observations, we suggest that the solution structure of the native hormone represents an inactive conformer and that a dynamic equilibrium exists between the C-terminally unfolded binding conformation and the inactive conformation of the RLF.


Assuntos
Insulina/química , Proteínas/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Reagentes de Ligações Cruzadas/metabolismo , Humanos , Insulina/síntese química , Insulina/metabolismo , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Subunidades Proteicas/síntese química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas/síntese química , Proteínas/metabolismo , Soluções
12.
Proteins ; 72(1): 333-43, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18214953

RESUMO

Electric fields generated in native proteins affect almost every aspect of protein function. We present a method that probes changes in the electric field at specific locations within a protein. The method utilizes the dependence of the amide (1)H and (15)N NMR chemical shifts on electric charges in proteins. Charges were introduced at different positions in the blue copper protein plastocyanin, by protonation of side chains or by substitution of the metal ion. It is found that the associated chemical shift perturbations (CSPs) stem mainly from long-range electric field effects caused by the change in the electric charge. It is demonstrated that the CSPs can be used to estimate the dielectric constant at different locations in the protein, estimate the nuclear shielding polarizability, or position charges in proteins.


Assuntos
Anabaena/química , Eletricidade , Plastocianina/química , Amidas , Sequência de Aminoácidos , Histidina/química , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Peptídeos/química , Prótons , Soluções , Eletricidade Estática
13.
J Am Chem Soc ; 130(26): 8460-70, 2008 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-18540585

RESUMO

A model describing conformational exchange of His 61 in plastocyanin from Anabaena variabilis is presented. A detailed picture of the exchange dynamics has been obtained using solution NMR relaxation measurements, chemical shift titrations, and structural information provided by a high-resolution crystal structure of the protein. A three-site model for chemical exchange that involves interconversion among the tautomeric and protonated forms of the histidine side chain with rates that are fast on the NMR chemical shift time scale can account for all of the data. In general, in the limit of fast exchange, it is not possible to obtain separate measures of chemical shift differences and populations of the participating states using NMR. However, we show here that when the data mentioned above are combined, it is possible to extract values of all of the parameters that characterize the exchange process, including rates, populations, and chemical shift changes, and to provide cross-validations that establish their accuracy. The methodology is generally applicable to the study of histidine side chain dynamics, which can play an important functional role in many protein systems.


Assuntos
Anabaena variabilis/química , Plastocianina/química , Histidina , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica
14.
Annu Rev Biophys ; 44: 53-75, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25747592

RESUMO

Electrostatics play an important role in many aspects of protein chemistry. However, the accurate determination of side chain proton affinity in proteins by experiment and theory remains challenging. In recent years the field of nuclear magnetic resonance spectroscopy has advanced the way that protonation states are measured, allowing researchers to examine electrostatic interactions at an unprecedented level of detail and accuracy. Experiments are now in place that follow pH-dependent (13)C and (15)N chemical shifts as spatially close as possible to the sites of protonation, allowing all titratable amino acid side chains to be probed sequence specifically. The strong and telling response of carefully selected reporter nuclei allows individual titration events to be monitored. At the same time, improved frameworks allow researchers to model multiple coupled protonation equilibria and to identify the underlying pH-dependent contributions to the chemical shifts.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Concentração de Íons de Hidrogênio , Prótons , Eletricidade Estática
15.
Curr Opin Struct Biol ; 24: 45-53, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24721452

RESUMO

Paramagnetic NMR spectroscopy has evolved rapidly in the last decade, and has shown to be a very useful tool for solving structures of protein-protein complexes. A major breakthrough has been the development of paramagnetic metal binding tags that can be attached specifically to the protein. These tags have greatly facilitated the use of anisotropic paramagnetic restraints such as pseudocontact shifts and residual dipolar couplings arising from paramagnetic self-alignment. Such restraints are particularly useful for the study of large protein complexes. This review focuses on the recent developments in structural characterization of protein-protein complexes using anisotropic paramagnetic NMR restraints.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Animais , Anisotropia , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Mapas de Interação de Proteínas , Proteínas/metabolismo
16.
J Mol Biol ; 425(22): 4353-65, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23856620

RESUMO

Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system. To understand the molecular interactions between putidaredoxin and cytochrome P450cam, we determined the structure of the complex both in solution and in the crystal state. Paramagnetic NMR spectroscopy using lanthanide tags yielded 446 structural restraints that were used to determine the solution structure. An ensemble of 10 structures with an RMSD of 1.3Å was obtained. The crystal structure of the complex was solved, showing a position of putidaredoxin that is identical with the one in the solution structure. The NMR data further demonstrate the presence of a minor state or set of states of the complex in solution, which is attributed to the presence of an encounter complex. The structure of the major state shows a small binding interface and a metal-to-metal distance of 16Å, with two pathways that provide strong electronic coupling of the redox centers. The interpretation of these results is discussed in the context of ET. The structure indicates that the ET rate can be much faster than the reported value, suggesting that the process may be gated.


Assuntos
Cânfora 5-Mono-Oxigenase/química , Ferredoxinas/química , Complexos Multiproteicos/química , Cânfora 5-Mono-Oxigenase/metabolismo , Cristalografia por Raios X , Ferredoxinas/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação Proteica
17.
J Med Chem ; 55(23): 10786-90, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-23145792

RESUMO

An efficient way to rapidly generate protein-ligand costructures based on solution-NMR using sparse NOE data combined with selective isotope labeling is presented. A docked model of the 27 kDa N-terminal ATPase domain of Hsp90 bound to a small molecule ligand was generated using only 21 intermolecular NOEs, which uniquely defined both the binding site and the orientation of the ligand. The approach can prove valuable for the early stages of fragment-based drug discovery.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Adenosina Trifosfatases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Ligantes
18.
Biochemistry ; 46(50): 14619-28, 2007 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-18020375

RESUMO

Exchange on the microsecond time scale between the protonated and deprotonated forms of His92 in the copper site of reduced plastocyanin from the cyanobacteria Anabaena variabilis was monitored using 15N NMR relaxation measurements. On the basis of the dependence of the kinetics on pH and phosphate buffer concentration, we propose a two-step model for the protonation of the copper site in agreement with previous crystallographic studies. It is shown that the proton transfer is the rate-limiting step in the reaction at low buffer concentrations, whereas at high buffer concentrations, another step becomes rate-limiting. We suggest that the latter step is a concerted dissociation of His92 from the Cu(I) ion and a 180 degrees rotation of the imidazole ring, which precede the protonation. The first-order rate constant for the dissociation of His92 from the Cu(I) ion is estimated to be 2.4 x 10(4) s(-1). Also, a cooperative effect of the protonation of the remote His61 on the protonation of His92 and the redox properties of the protein was investigated by substituting His61 with asparagine. The mutation causes a modest change in both the pKa value of His92 and the redox potential of the protein.


Assuntos
Proteínas de Bactérias/química , Plastocianina/química , Anabaena/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cobre/química , Cobre/metabolismo , Histidina/química , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Ressonância Magnética Nuclear Biomolecular , Plastocianina/metabolismo , Prótons
19.
Magn Reson Chem ; 44(8): 761-9, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16705625

RESUMO

Two methods for estimating the microsecond-millisecond dynamics in proteins from only two 15N relaxation parameters at one magnetic field strength are investigated. Thus, the chemical exchange contribution, R(ex), to the transversal relaxation rate, which contains the dynamics information, is evaluated by two methods: (i) one in which the R(ex) term is derived from the 15N R1 and R2 relaxation rates alone, and (ii) one in which it is obtained from the transversal dipole-chemical shift anisotropy (CSA) cross-correlation rate, eta(xy), and the R2 rate. Since the R1, R2, and eta(xy) experiments are fast and sensitive, both methods are attractive in studies where large amounts of dynamical information are required. However, both methods are liable to effects that can compromise the estimation of the R(ex) terms. In the R2/R1 method, internal ps-ns dynamics and rotational anisotropy can interfere with the determination of R(ex), while in the R2/eta(xy) method it can be affected by variations in the 15N chemical shift anisotropy. Here, the applicability of the two methods is investigated using plastocyanin from Anabaena variabilis as an example, and the quality of the obtained R(ex) terms is evaluated both theoretically and experimentally. It is found that the R2/R1 method gives reliable R(ex) terms if the protein is relatively rigid and tumbles fast and nearly isotropically in solution, as for instance plastocyanin, and is preferable in such cases. In contrast, the R2/eta(xy) method gives better results if the protein is flexible or highly non-spherical and can be used for such proteins, if the sequential variation in the 15N chemical shift anisotropy is negligible. For exchange terms <1 s(-1) neither method is reliable.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Aminoácidos/química , Anabaena variabilis/química , Anisotropia , Isótopos de Nitrogênio/análise , Plastocianina/química
20.
Magn Reson Chem ; 44(3): 294-301, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16477687

RESUMO

The utility of pseudocontact shifts in the structure refinement of metalloproteins has been evaluated using a native, paramagnetic Cu(2+) metalloprotein, plastocyanin from Anabaena variabilis (A.v.), as a model protein. First, the possibility of detecting signals of nuclei spatially close to the paramagnetic metal ion is investigated using the WEFT pulse sequence in combination with the conventional TOCSY and (1)H-(15)N HSQC sequences. Second, the importance of the electrical charge of the metal ion for the determination of correct pseudocontact shifts from the obtained chemical shifts is evaluated. Thus, using both the Cu(+) plastocyanin and Cd(2+)-substituted plastocyanin as the diamagnetic references, it is found that the Cd(2+)-substituted protein with the same electrical charge of the metal ion as the paramagnetic Cu(2+) plastocyanin provides the most appropriate diamagnetic reference signals. Third, it is found that reliable pseudocontact shifts cannot be obtained from the chemical shifts of the (15)N nuclei in plastocyanin, most likely because these shifts are highly dependent on even minor differences in the structure of the paramagnetic and diamagnetic proteins. Finally, the quality of the obtained (1)H pseudocontact shifts, as well as the possibility of improving the accuracy of the obtained structure, is demonstrated by incorporating the shifts as restraints in a refinement of the solution structure of A.v. plastocyanin. It is found that incorporation of the pseudocontact shifts enhances the precision of the structure in regions with only few NOE restraints and improves the accuracy of the overall structure.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Plastocianina/química , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química
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