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1.
Proc Natl Acad Sci U S A ; 117(34): 20576-20585, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32788352

RESUMO

Temperate bacteriophages can enter one of two life cycles following infection of a sensitive host: the lysogenic or the lytic life cycle. The choice between the two alternative life cycles is dependent upon a tight regulation of promoters and their cognate regulatory proteins within the phage genome. We investigated the genetic switch of TP901-1, a bacteriophage of Lactococcus lactis, controlled by the CI repressor and the modulator of repression (MOR) antirepressor and their interactions with DNA. We determined the solution structure of MOR, and we solved the crystal structure of MOR in complex with the N-terminal domain of CI, revealing the structural basis of MOR inhibition of CI binding to the DNA operator sites. 15N NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion and rotating frame R1ρ measurements demonstrate that MOR displays molecular recognition dynamics on two different time scales involving a repacking of aromatic residues at the interface with CI. Mutations in the CI:MOR binding interface impair complex formation in vitro, and when introduced in vivo, the bacteriophage switch is unable to choose the lytic life cycle showing that the CI:MOR complex is essential for proper functioning of the genetic switch. On the basis of sequence alignments, we show that the structural features of the MOR:CI complex are likely conserved among a larger family of bacteriophages from human pathogens implicated in transfer of antibiotic resistance.


Assuntos
Bacteriófagos/fisiologia , Lisogenia , Proteínas Repressoras/fisiologia , Proteínas Virais Reguladoras e Acessórias/fisiologia , Genoma Bacteriano , Interações Hospedeiro-Patógeno , Cinética , Lactococcus lactis/virologia , Simulação de Dinâmica Molecular , Regiões Operadoras Genéticas , Conformação Proteica , Proteínas Repressoras/química , Proteínas Virais Reguladoras e Acessórias/química
2.
J Struct Biol ; 209(3): 107434, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31846718

RESUMO

In bacteria, nucleoid associated proteins (NAPs) take part in active chromosome organization by supercoil management, three-dimensional DNA looping and direct transcriptional control. Mycobacterial integration host factor (mIHF, rv1388) is a NAP restricted to Actinobacteria and essential for survival of the human pathogen Mycobacterium tuberculosis. We show in vitro that DNA binding by mIHF strongly stabilizes the protein and increases its melting temperature. The structure obtained by Nuclear Magnetic Resonance (NMR) spectroscopy characterizes mIHF as a globular protein with a protruding alpha helix and a disordered N-terminus, similar to Streptomyces coelicolor IHF (sIHF). NMR revealed no residues of high flexibility, suggesting that mIHF is a rigid protein overall that does not undergo structural rearrangements. We show that mIHF only binds to double stranded DNA in solution, through two DNA binding sites (DBSs) similar to those identified in the X-ray structure of sIHF. According to Atomic Force Microscopy, mIHF is able to introduce left-handed loops of ca. 100 nm size (~300 bp) in supercoiled cosmids, thereby unwinding and relaxing the DNA.


Assuntos
Proteínas de Ligação a DNA/ultraestrutura , Fatores Hospedeiros de Integração/ultraestrutura , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Sítios de Ligação/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Fatores Hospedeiros de Integração/genética , Espectroscopia de Ressonância Magnética , Microscopia de Força Atômica , Mycobacterium tuberculosis/patogenicidade , Conformação Proteica em alfa-Hélice/genética , Streptomyces coelicolor/genética , Tuberculose/genética
3.
Proc Natl Acad Sci U S A ; 113(33): 9187-92, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27489348

RESUMO

Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of (1)H-(1)H proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination. Additionally, we find that narrower proton resonance lines, longer coherence lifetimes, and improved magnetization transfer offset the reduced sample size at 100-kHz spinning and above. Less than 2 weeks of experiment time and a single 0.5-mg sample was sufficient for the acquisition of all data necessary for backbone and side-chain resonance assignment and unsupervised structure determination. We expect the technique to pave the way for atomic-resolution structure analysis applicable to a wide range of proteins.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas/química , Dobramento de Proteína , Prótons
4.
J Biol Chem ; 290(33): 20527-40, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26149686

RESUMO

Cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic agent for Parkinson disease. As such, there has been great interest in studying its mode of action, which remains unknown. The three-dimensional crystal structure of the N terminus (residues 9-107) of CDNF has been determined, but there have been no published structural studies on the full-length protein due to proteolysis of its C-terminal domain, which is considered intrinsically disordered. An improved purification protocol enabled us to obtain active full-length CDNF and to determine its three-dimensional structure in solution. CDNF contains two well folded domains (residues 10-100 and 111-157) that are linked by a loop of intermediate flexibility. We identified two surface patches on the N-terminal domain that were characterized by increased conformational dynamics that should allow them to embrace active sites. One of these patches is formed by residues Ser-33, Leu-34, Ala-66, Lys-68, Ile-69, Leu-70, Ser-71, and Glu-72. The other includes a flexibly disordered N-terminal tail (residues 1-9), followed by the N-terminal portion of α-helix 1 (residues Cys-11, Glu-12, Val-13, Lys-15, and Glu-16) and residue Glu-88. The surface of the C-terminal domain contains two conserved active sites, which have previously been identified in mesencephalic astrocyte-derived neurotrophic factor, a CDNF paralog, which corresponds to its intracellular mode of action. We also showed that CDNF was able to protect dopaminergic neurons against injury caused by α-synuclein oligomers. This advises its use against physiological damages caused by α-synuclein oligomers, as observed in Parkinson disease and several other neurodegenerative diseases.


Assuntos
Biopolímeros/metabolismo , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/fisiologia , Fármacos Neuroprotetores , alfa-Sinucleína/metabolismo , Animais , Linhagem Celular , Cristalografia por Raios X , Humanos , Camundongos , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Relação Estrutura-Atividade
5.
J Biomol NMR ; 64(1): 27-37, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26614488

RESUMO

Nuclear magnetic resonance spectroscopy (NMR) can provide a great deal of information about structure and dynamics of biomolecules. The quality of an NMR structure strongly depends on the number of experimental observables and on their accurate conversion into geometric restraints. When distance restraints are derived from nuclear Overhauser effect spectroscopy (NOESY), stereo-specific assignments of prochiral atoms can contribute significantly to the accuracy of NMR structures of proteins and nucleic acids. Here we introduce a series of NOESY-based pulse sequences that can assist in the assignment of chiral CHD methylene protons in random fractionally deuterated proteins. Partial deuteration suppresses spin-diffusion between the two protons of CH2 groups that normally impedes the distinction of cross-relaxation networks for these two protons in NOESY spectra. Three and four-dimensional spectra allow one to distinguish cross-relaxation pathways involving either of the two methylene protons so that one can obtain stereospecific assignments. In addition, the analysis provides a large number of stereospecific distance restraints. Non-uniform sampling was used to ensure optimal signal resolution in 4D spectra and reduce ambiguities of the assignments. Automatic assignment procedures were modified for efficient and accurate stereospecific assignments during automated structure calculations based on 3D spectra. The protocol was applied to calcium-loaded calbindin D9k. A large number of stereospecific assignments lead to a significant improvement of the accuracy of the structure.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Modelos Moleculares , Conformação Molecular , Proteína G de Ligação ao Cálcio S100/química
6.
J Biomol NMR ; 62(4): 473-80, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25917899

RESUMO

UNIO is a comprehensive software suite for protein NMR structure determination that enables full automation of all NMR data analysis steps involved--including signal identification in NMR spectra, sequence-specific backbone and side-chain resonance assignment, NOE assignment and structure calculation. Within the framework of the second round of the community-wide stringent blind NMR structure determination challenge (CASD-NMR 2), we participated in two categories of CASD-NMR 2, namely using either raw NMR spectra or unrefined NOE peak lists as input. A total of 15 resulting NMR structure bundles were submitted for 9 out of 10 blind protein targets. All submitted UNIO structures accurately coincided with the corresponding blind targets as documented by an average backbone root mean-square deviation to the reference proteins of only 1.2 Å. Also, the precision of the UNIO structure bundles was virtually identical to the ensemble of reference structures. By assessing the quality of all UNIO structures submitted to the two categories, we find throughout that only the UNIO-ATNOS/CANDID approach using raw NMR spectra consistently yielded structure bundles of high quality for direct deposition in the Protein Data Bank. In conclusion, the results obtained in CASD-NMR 2 are another vital proof for robust, accurate and unsupervised NMR data analysis by UNIO for real-world applications.


Assuntos
Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas/química , Software , Ressonância Magnética Nuclear Biomolecular/métodos
7.
J Biomol NMR ; 61(1): 47-53, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25428764

RESUMO

A standard set of three APSY-NMR experiments has been used in daily practice to obtain polypeptide backbone NMR assignments in globular proteins with sizes up to about 150 residues, which had been identified as targets for structure determination by the Joint Center for Structural Genomics (JCSG) under the auspices of the Protein Structure Initiative (PSI). In a representative sample of 30 proteins, initial fully automated data analysis with the software UNIO-MATCH-2014 yielded complete or partial assignments for over 90 % of the residues. For most proteins the APSY data acquisition was completed in less than 30 h. The results of the automated procedure provided a basis for efficient interactive validation and extension to near-completion of the assignments by reference to the same 3D heteronuclear-resolved [(1)H,(1)H]-NOESY spectra that were subsequently used for the collection of conformational constraints. High-quality structures were obtained for all 30 proteins, using the J-UNIO protocol, which includes extensive automation of NMR structure determination.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Software , Estrutura Terciária de Proteína
8.
J Biomol NMR ; 62(3): 253-61, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26078089

RESUMO

Here we introduce a new pulse sequence for resonance assignment that halves the number of data sets required for sequential linking by directly correlating sequential amide resonances in a single diagonal-free spectrum. The method is demonstrated with both microcrystalline and sedimented deuterated proteins spinning at 60 and 111 kHz, and a fully protonated microcrystalline protein spinning at 111 kHz, with as little as 0.5 mg protein sample. We find that amide signals have a low chance of ambiguous linkage, which is further improved by linking in both forward and backward directions. The spectra obtained are amenable to automated resonance assignment using general-purpose software such as UNIO-MATCH.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Prótons
9.
Proc Natl Acad Sci U S A ; 109(28): 11095-100, 2012 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-22723345

RESUMO

We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with (1)H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of (15)N and (13)C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu(+) (diamagnetic) or Cu(2+) (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to (1)H-(1)H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.


Assuntos
Carbono/química , Espectroscopia de Ressonância Magnética/métodos , Metaloproteínas/química , Nitrogênio/química , Catálise , Domínio Catalítico , Cobre/química , Cristalização , Hidrogênio/química , Modelos Moleculares , Conformação Molecular , Distribuição Normal , Ressonância Magnética Nuclear Biomolecular/métodos , Prótons
10.
J Am Chem Soc ; 136(35): 12489-97, 2014 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-25102442

RESUMO

Using a set of six (1)H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5-30 kDa proteins. The approach relies on perdeuteration, amide (2)H/(1)H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary (13)C/(15)N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.


Assuntos
Hidrogênio/análise , Ressonância Magnética Nuclear Biomolecular/métodos , Prótons , Isótopos de Carbono/análise , Medição da Troca de Deutério , Modelos Moleculares , Isótopos de Nitrogênio/análise , Proteínas/química
11.
Nat Chem Biol ; 8(10): 855-61, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22941047

RESUMO

Among bacterial toxin-antitoxin systems, to date no antitoxin has been identified that functions by cleaving toxin mRNA. Here we show that YjdO (renamed GhoT) is a membrane lytic peptide that causes ghost cell formation (lysed cells with damaged membranes) and increases persistence (persister cells are tolerant to antibiotics without undergoing genetic change). GhoT is part of a new toxin-antitoxin system with YjdK (renamed GhoS) because in vitro RNA degradation studies, quantitative real-time reverse-transcription PCR and whole-transcriptome studies revealed that GhoS masks GhoT toxicity by cleaving specifically yjdO (ghoT) mRNA. Alanine substitutions showed that Arg28 is important for GhoS activity, and RNA sequencing indicated that the GhoS cleavage site is rich in U and A. The NMR structure of GhoS indicates it is related to the CRISPR-associated-2 RNase, and GhoS is a monomer. Hence, GhoT-GhoS is to our knowledge the first type V toxin-antitoxin system where a protein antitoxin inhibits the toxin by cleaving specifically its mRNA.


Assuntos
Antitoxinas/genética , Toxinas Bacterianas/genética , RNA Mensageiro/genética , Antitoxinas/química , Antitoxinas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Biofilmes , Hidrólise , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Sci Adv ; 10(31): eaax2323, 2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-39093972

RESUMO

The nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 encapsidates the viral genome and is essential for viral function. The central disordered domain comprises a serine-arginine-rich (SR) region that is hyperphosphorylated in infected cells. This modification regulates function, although mechanistic details remain unknown. We use nuclear magnetic resonance to follow structural changes occurring during hyperphosphorylation by serine arginine protein kinase 1, glycogen synthase kinase 3, and casein kinase 1, that abolishes interaction with RNA. When eight approximately uniformly distributed sites have been phosphorylated, the SR domain binds the same interface as single-stranded RNA, resulting in complete inhibition of RNA binding. Phosphorylation by protein kinase A does not prevent RNA binding, indicating that the pattern resulting from physiologically relevant kinases is specific for inhibition. Long-range contacts between the RNA binding, linker, and dimerization domains are abrogated, phenomena possibly related to genome packaging and unpackaging. This study provides insight into the recruitment of specific host kinases to regulate viral function.


Assuntos
Proteínas do Nucleocapsídeo de Coronavírus , Ligação Proteica , RNA Viral , SARS-CoV-2 , Fosforilação , SARS-CoV-2/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/química , Humanos , RNA Viral/metabolismo , RNA Viral/química , Conformação Proteica , COVID-19/virologia , COVID-19/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/química , Modelos Moleculares , Sítios de Ligação , Fosfoproteínas
13.
Q Rev Biophys ; 44(3): 257-309, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21411039

RESUMO

Around half of all protein structures solved nowadays using solution-state nuclear magnetic resonance (NMR) spectroscopy have been because of automated data analysis. The pervasiveness of computational approaches in general hides, however, a more nuanced view in which the full variety and richness of the field appears. This review is structured around a comparison of methods associated with three NMR observables: classical nuclear Overhauser effect (NOE) constraint gathering in contrast with more recent chemical shift and residual dipole coupling (RDC) based protocols. In each case, the emphasis is placed on the latest research, covering mainly the past 5 years. By describing both general concepts and representative programs, the objective is to map out a field in which--through the very profusion of approaches--it is all too easy to lose one's bearings.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Automação , Humanos , Reprodutibilidade dos Testes
14.
J Biol Chem ; 286(2): 1364-73, 2011 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-20929865

RESUMO

Ubiquitin-binding domains (UBDs) provide specificity to the ubiquitin system, which is also involved in translesion synthesis (TLS) in eukaryotic cells. Upon DNA damage, the UBDs (UBM domains) of polymerase iota (Pol ι) interact with ubiquitinated proliferating cell nuclear antigen to regulate the interchange between processive DNA polymerases and TLS. We report a biophysical analysis and solution structures of the two conserved UBM domains located in the C-terminal tail of murine Pol ι in complex with ubiquitin. The 35-amino acid core folds into a helix-turn-helix motif, which belongs to a novel domain fold. Similar to other UBDs, UBMs bind to ubiquitin on the hydrophobic surface delineated by Leu-8, Ile-44, and Val-70, however, slightly shifted toward the C terminus. In addition, UBMs also use electrostatic interactions to stabilize binding. NMR and fluorescence spectroscopy measurements revealed that UBMs bind monoubiquitin, and Lys-63- but not Lys-48-linked chains. Importantly, these biophysical data are supported by functional studies. Indeed, yeast cells expressing ubiquitin mutants specifically defective for UBM binding are viable but sensitive to DNA damaging conditions that require TLS for repair.


Assuntos
Dano ao DNA/fisiologia , DNA Polimerase Dirigida por DNA , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Animais , Sítios de Ligação/fisiologia , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae , DNA Polimerase iota
15.
J Am Chem Soc ; 134(36): 14730-3, 2012 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-22916960

RESUMO

Pseudocontact shifts (PCSs) arise in paramagnetic systems in which the susceptibility tensor is anisotropic. PCSs depend upon the distance from the paramagnetic center and the position relative to the susceptibility tensor, and they can be used as structural restraints in protein structure determination. We show that the use of (1)H-detected solid-state correlations provides facile and rapid detection and assignment of site-specific PCSs, including resolved (1)H PCSs, in a large metalloprotein, Co(2+)-substituted superoxide dismutase (Co(2+)-SOD). With only 3 mg of sample and a small set of experiments, several hundred PCSs were measured and assigned, and these PCSs were subsequently used in combination with (1)H-(1)H distance and dihedral angle restraints to determine the protein backbone geometry with a precision paralleling those of state-of-the-art liquid-state determinations of diamagnetic proteins, including a well-defined active site.


Assuntos
Cobalto/química , Espectroscopia de Ressonância Magnética/normas , Metaloproteínas/química , Prótons , Superóxido Dismutase/química , Modelos Moleculares , Padrões de Referência , Superóxido Dismutase/metabolismo
16.
J Biomol NMR ; 53(4): 341-54, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22752932

RESUMO

The J-UNIO (JCSG protocol using the software UNIO) procedure for automated protein structure determination by NMR in solution is introduced. In the present implementation, J-UNIO makes use of APSY-NMR spectroscopy, 3D heteronuclear-resolved [(1)H,(1)H]-NOESY experiments, and the software UNIO. Applications with proteins from the JCSG target list with sizes up to 150 residues showed that the procedure is highly robust and efficient. In all instances the correct polypeptide fold was obtained in the first round of automated data analysis and structure calculation. After interactive validation of the data obtained from the automated routine, the quality of the final structures was comparable to results from interactive structure determination. Special advantages are that the NMR data have been recorded with 6-10 days of instrument time per protein, that there is only a single step of chemical shift adjustments to relate the backbone signals in the APSY-NMR spectra with the corresponding backbone signals in the NOESY spectra, and that the NOE-based amino acid side chain chemical shift assignments are automatically focused on those residues that are heavily weighted in the structure calculation. The individual working steps of J-UNIO are illustrated with the structure determination of the protein YP_926445.1 from Shewanella amazonensis, and the results obtained with 17 JCSG targets are critically evaluated.


Assuntos
Proteínas/química , Software , Sequência de Aminoácidos , Proteínas de Bactérias/química , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Shewanella/química , Soluções
17.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 66(Pt 10): 1393-405, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20944236

RESUMO

The JCSG has recently developed a protocol for systematic comparisons of high-quality crystal and NMR structures of proteins. In this paper, the extent to which this approach can provide function-related information on the two functionally annotated proteins TM1081, a Thermotoga maritima anti-σ factor antagonist, and A2LD1 (gi:13879369), a mouse γ-glutamylamine cyclotransferase, is explored. The NMR structures of the two proteins have been determined in solution at 313 and 298 K, respectively, using the current JCSG protocol based on the software package UNIO for extensive automation. The corresponding crystal structures were solved by the JCSG at 100 K and 1.6 Šresolution and at 100 K and 1.9 Šresolution, respectively. The NMR and crystal structures of the two proteins share the same overall molecular architectures. However, the precision of the structure determination along the amino-acid sequence varies over a significantly wider range in the NMR structures than in the crystal structures. Thereby, in each of the two NMR structures about 65% of the residues have displacements below the average and in both proteins the less well ordered residues include large parts of the active sites, in addition to some highly solvent-exposed surface areas. Whereas the latter show increased disorder in the crystal and in solution, the active-site regions display increased displacements only in the NMR structures, where they undergo local conformational exchange on the millisecond time scale that appears to be frozen in the crystals. These observations suggest that a search for molecular regions showing increased structural disorder and slow dynamic processes in solution while being well ordered in the corresponding crystal structure might be a valid initial step in the challenge of identifying putative active sites in functionally unannotated proteins with known three-dimensional structure.


Assuntos
Proteínas de Bactérias/análise , Domínio Catalítico , Thermotoga maritima/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Isomerismo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas
18.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 66(Pt 10): 1381-92, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20944235

RESUMO

The NMR structures of the TM1112 and TM1367 proteins from Thermotoga maritima in solution at 298 K were determined following a new protocol which uses the software package UNIO for extensive automation. The results obtained with this novel procedure were evaluated by comparison with the crystal structures solved by the JCSG at 100 K to 1.83 and 1.90 Šresolution, respectively. In addition, the TM1112 solution structure was compared with an NMR structure solved by the NESG using a conventional largely interactive methodology. For both proteins, the newly determined NMR structure could be superimposed with the crystal structure with r.m.s.d. values of <1.0 Šfor the backbone heavy atoms, which provided a starting platform to investigate local structure variations, which may arise from either the methods used or from the different chemical environments in solution and in the crystal. Thereby, these comparative studies were further explored with the use of reference NMR and crystal structures, which were computed using the NMR software with input of upper-limit distance constraints derived from the molecular models that represent the results of structure determination by NMR and by X-ray diffraction, respectively. The results thus obtained show that NMR structure calculations with the new automated UNIO software used by the JCSG compare favorably with those from a more labor-intensive and time-intensive interactive procedure. An intriguing observation is that the `bundles' of two TM1112 or three TM1367 molecules in the asymmetric unit of the crystal structures mimic the behavior of the bundles of 20 conformers used to represent the NMR solution structures when comparing global r.m.s.d. values calculated either for the polypeptide backbone, the core residues with solvent accessibility below 15% or all heavy atoms.


Assuntos
Proteínas de Bactérias/química , Thermotoga maritima/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína
19.
Magn Reson Chem ; 48(9): 727-33, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20648569

RESUMO

Unambiguous identification of individual metabolites present in complex mixtures such as biofluids constitutes a crucial prerequisite for quantitative metabolomics, toward better understanding of biochemical processes in living systems. Increasing the dimensionality of a given NMR correlation experiment is the natural solution for resolving spectral overlap. However, in the context of metabolites, natural abundance acquisition of (1)H and (13)C NMR data virtually excludes the use of higher dimensional NMR experiments (3D, 4D, etc.) that would require unrealistically long acquisition times. Here, we introduce projection NMR techniques for studies of complex mixtures, and we show how discrete sets of projection spectra from higher dimensional NMR experiments are obtained in a reasonable time frame, in order to capture essential information necessary to resolve assignment ambiguities caused by signal overlap in conventional 2D NMR spectra. We determine optimal projection angles where given metabolite resonances will have the least overlap, to obtain distinct metabolite assignment in complex mixtures. The method is demonstrated for a model mixture composition made of ornithine, putrescine and arginine for which acquisition of a single 2D projection of a 3D (1)H-(13)C TOCSY-HSQC spectrum allows to disentangle the metabolite signals and to access to complete profiling of this model mixture in the targeted 2D projection plane.


Assuntos
Arginina/química , Hipuratos/química , Ornitina/química , Putrescina/química , Arginina/metabolismo , Hipuratos/metabolismo , Espectroscopia de Ressonância Magnética/normas , Estrutura Molecular , Ornitina/metabolismo , Putrescina/metabolismo , Padrões de Referência
20.
Dev Cell ; 50(4): 494-508.e11, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31430451

RESUMO

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the µ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. µ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with µ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.


Assuntos
Complexo 2 de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/genética , Endocitose/genética , Nexinas de Classificação/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Clatrina/genética , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/genética , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/genética , Invaginações Revestidas da Membrana Celular/metabolismo , Humanos , Fosforilação/genética , Ligação Proteica/genética
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