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1.
Biochemistry ; 59(8): 964-969, 2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-32032490

RESUMO

KLHL-12 is a substrate specific adapter protein for a Cul3-Ring ligase complex. It is a member of the Kelch ß-propeller domain subclass of Cullin-Ring substrate recognition domains. This E3 ubiquitin ligase complex has many activities, including acting as a negative regulator of the Wnt signaling pathway by mediating ubiquitination and subsequent proteolysis of Dvl3/Dsh3. KLHL-12 is also known to mediate the polyubiquitination of the dopamine D4 receptor (D4.2), the ubiquitination of KHSRP, a protein that is involved in IRES translation, and also the ubiquitination of Sec31, which is involved in endoplasmic reticulum-Golgi transport by regulating the size of COPII coats. Earlier studies broadly defined the substrate binding regions for D4.2 and Dvl3/Dsh3 to KLHL-12. We tested several peptides from these regions and succeeded in identifying a short peptide that bound to KLHL-12 with low micromolar affinity. To better understand the sequence specificity of this peptide, we used alanine substitutions to map the important residues and obtained an X-ray structure of this peptide bound to KLHL-12. This structure and our peptide affinity measurements suggest a sequence motif for peptides that bind to the top face of KLHL-12. Understanding this binding site on KLHL-12 may contribute to efforts to find small molecule ligands that can either directly inhibit the degradation of substrate proteins or be used in targeted protein degradation strategies using PROTACs.

2.
Protein Eng Des Sel ; 31(5): 181-190, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29992271

RESUMO

Computationally designed transmembrane α-helical peptides (CHAMP) have been used to compete for helix-helix interactions within the membrane, enabling the ability to probe the activation of the integrins αIIbß3 and αvß3. Here, this method is extended towards the design of CHAMP peptides that inhibit the association of the α5ß1 transmembrane (TM) domains, targeting the Ala-X3-Gly motif within α5. Our previous design algorithm was performed alongside a new workflow implemented within the widely used Rosetta molecular modeling suite. Peptides from each computational approach activated integrin α5ß1 but not αVß3 in human endothelial cells. Two CHAMP peptides were shown to directly associate with an α5 TM domain peptide in detergent micelles to a similar degree as a ß1 TM peptide does. By solution-state nuclear magnetic resonance, one of these CHAMP peptides was shown to bind primarily the integrin ß1 TM domain, which itself has a Gly-X3-Gly motif. The second peptide associated modestly with both α5 and ß1 constructs, with slight preference for α5. Although the design goal was not fully realized, this work characterizes novel CHAMP peptides activating α5ß1 that can serve as useful reagents for probing integrin biology.


Assuntos
Membrana Celular/metabolismo , Projeto Auxiliado por Computador , Integrina alfa5beta1/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Desenho de Drogas , Humanos , Micelas , Peptídeos/metabolismo , Conformação Proteica em alfa-Hélice , Domínios Proteicos
3.
Biochemistry ; 56(41): 5481-5484, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-28980804

RESUMO

There remains a need for new non-ionic detergents that are suitable for use in biochemical and biophysical studies of membrane proteins. Here we explore the properties of n-dodecyl-ß-melibioside (ß-DDMB) micelles as a medium for membrane proteins. Melibiose is d-galactose-α(1→6)-d-glucose. Light scattering showed the ß-DDMB micelle to be roughly 30 kDa smaller than micelles formed by the commonly used n-dodecyl-ß-maltoside (ß-DDM). ß-DDMB stabilized diacylglycerol kinase (DAGK) against thermal inactivation. Moreover, activity assays conducted using aliquots of DAGK purified into ß-DDMB yielded activities that were 40% higher than those of DAGK purified into ß-DDM. ß-DDMB yielded similar or better TROSY-HSQC NMR spectra for two single-pass membrane proteins and the tetraspan membrane protein peripheral myelin protein 22. ß-DDMB appears be a useful addition to the toolbox of non-ionic detergents available for membrane protein research.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Detergentes/química , Diacilglicerol Quinase/metabolismo , Dissacarídeos/química , Proteínas de Escherichia coli/metabolismo , Glicolipídeos/química , Proteínas da Mielina/metabolismo , Receptor Notch1/metabolismo , Precursor de Proteína beta-Amiloide/química , Detergentes/síntese química , Diacilglicerol Quinase/química , Dissacarídeos/síntese química , Difusão Dinâmica da Luz , Estabilidade Enzimática , Proteínas de Escherichia coli/química , Glucosídeos/química , Glicolipídeos/síntese química , Temperatura Alta/efeitos adversos , Humanos , Micelas , Proteínas da Mielina/química , Ressonância Magnética Nuclear Biomolecular , Tamanho da Partícula , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Receptor Notch1/química
4.
Development ; 144(22): 4148-4158, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28993400

RESUMO

Kidney collecting system development requires integrin-dependent cell-extracellular matrix interactions. Integrins are heterodimeric transmembrane receptors consisting of α and ß subunits; crucial integrins in the kidney collecting system express the ß1 subunit. The ß1 cytoplasmic tail has two NPxY motifs that mediate functions by binding to cytoplasmic signaling and scaffolding molecules. Talins, scaffolding proteins that bind to the membrane proximal NPxY motif, are proposed to activate integrins and to link them to the actin cytoskeleton. We have defined the role of talin binding to the ß1 proximal NPxY motif in the developing kidney collecting system in mice that selectively express a Y-to-A mutation in this motif. The mice developed a hypoplastic dysplastic collecting system. Collecting duct cells expressing this mutation had moderate abnormalities in cell adhesion, migration, proliferation and growth factor-dependent signaling. In contrast, mice lacking talins in the developing ureteric bud developed kidney agenesis and collecting duct cells had severe cytoskeletal, adhesion and polarity defects. Thus, talins are essential for kidney collecting duct development through mechanisms that extend beyond those requiring binding to the ß1 integrin subunit NPxY motif.


Assuntos
Integrina beta1/metabolismo , Morfogênese , Talina/metabolismo , Ureter/citologia , Ureter/embriologia , Junções Aderentes/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Adesão Celular , Membrana Celular/metabolismo , Polaridade Celular , Regulação da Expressão Gênica no Desenvolvimento , Integrina beta1/química , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/embriologia , Camundongos Endogâmicos C57BL , Mutação/genética , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo , Ureter/metabolismo
5.
Molecules ; 22(8)2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28809779

RESUMO

A large portion of proteins in living organisms are membrane proteins which play critical roles in the biology of the cell, from maintenance of the biological membrane integrity to communication of cells with their surroundings. To understand their mechanism of action, structural information is essential. Nevertheless, structure determination of transmembrane proteins is still a challenging area, even though recently the number of deposited structures of membrane proteins in the PDB has rapidly increased thanks to the efforts using X-ray crystallography, electron microscopy, and solid and solution nuclear magnetic resonance (NMR) technology. Among these technologies, solution NMR is a powerful tool for studying protein-protein, protein-ligand interactions and protein dynamics at a wide range of time scales as well as structure determination of membrane proteins. This review provides general and useful guideline for membrane protein sample preparation and the choice of membrane-mimetic media, which are the key step for successful structural analysis. Furthermore, this review provides an opportunity to look at recent applications of solution NMR to structural studies on α-helical membrane proteins through some success stories.


Assuntos
Proteínas de Membrana/química , Animais , Membrana Celular/química , Cristalografia por Raios X , Humanos , Ligantes , Microscopia Eletrônica , Conformação Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica
6.
Sci Adv ; 3(4): e1602794, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28439555

RESUMO

γ-Secretase cleavage of the Notch receptor transmembrane domain is a critical signaling event for various cellular processes. Efforts to develop inhibitors of γ-secretase cleavage of the amyloid-ß precursor C99 protein as potential Alzheimer's disease therapeutics have been confounded by toxicity resulting from the inhibition of normal cleavage of Notch. We present biochemical and structural data for the combined transmembrane and juxtamembrane Notch domains (Notch-TMD) that illuminate Notch signaling and that can be compared and contrasted with the corresponding traits of C99. The Notch-TMD and C99 have very different conformations, adapt differently to changes in model membrane hydrophobic span, and exhibit different cholesterol-binding properties. These differences may be exploited in the design of agents that inhibit cleavage of C99 while allowing Notch cleavage.


Assuntos
Precursor de Proteína beta-Amiloide/química , Modelos Moleculares , Receptores Notch/química , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Receptores Notch/genética , Receptores Notch/metabolismo
7.
Elife ; 52016 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-27929375

RESUMO

Integrins are transmembrane receptors composed of α and ß subunits. Although most integrins contain ß1, canonical activation mechanisms are based on studies of the platelet integrin, αIIbß3. Its inactive conformation is characterized by the association of the αIIb transmembrane and cytosolic domain (TM/CT) with a tilted ß3 TM/CT that leads to activation when disrupted. We show significant structural differences between ß1 and ß3 TM/CT in bicelles. Moreover, the 'snorkeling' lysine at the TM/CT interface of ß subunits, previously proposed to regulate αIIbß3 activation by ion pairing with nearby lipids, plays opposite roles in ß1 and ß3 integrin function and in neither case is responsible for TM tilt. A range of affinities from almost no interaction to the relatively high avidity that characterizes αIIbß3 is seen between various α subunits and ß1 TM/CTs. The αIIbß3-based canonical model for the roles of the TM/CT in integrin activation and function clearly does not extend to all mammalian integrins.


Assuntos
Células Epiteliais/fisiologia , Integrina alfa1/metabolismo , Integrina beta1/metabolismo , Integrina beta3/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Multimerização Proteica , Substituição de Aminoácidos , Adesão Celular , Células Cultivadas , Células Epiteliais/química , Humanos , Integrina alfa1/química , Integrina beta1/química , Integrina beta1/genética , Integrina beta3/química , Integrina beta3/genética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/química , Ligação Proteica
8.
Biophys J ; 110(11): 2475-2485, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27276265

RESUMO

Caveolins mediate the formation of caveolae, which are small omega-shaped membrane invaginations involved in a variety of cellular processes. There are three caveolin isoforms, the third of which (Cav3) is expressed in smooth and skeletal muscles. Mutations in Cav3 cause a variety of human muscular diseases. In this work, we characterize the secondary structure, dynamics, and topology of the monomeric form of the full-length lipidated protein. Cav3 consists of a series of membrane-embedded or surface-associated helical elements connected by extramembrane connecting loops or disordered domains. Our results also reveal that the N-terminal domain undergoes a large scale pH-mediated topological rearrangement between soluble and membrane-anchored forms. Considering that roughly one-third of pathogenic mutations in Cav3 influence charged residues located in this domain, we hypothesize that this transition is likely to be relevant to the molecular basis of Cav3-linked diseases. These results provide insight into the structure of Cav3 and set the stage for mechanistic investigations of the effects of pathogenic mutations.


Assuntos
Caveolina 3/metabolismo , Concentração de Íons de Hidrogênio , Sequência de Aminoácidos , Caveolina 3/genética , Dicroísmo Circular , Humanos , Membranas Artificiais , Micelas , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Fosfatidilgliceróis/química , Estrutura Secundária de Proteína , Solubilidade , Soluções
9.
Biochemistry ; 54(23): 3565-8, 2015 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-26023825

RESUMO

The Notch signaling pathway is critical in development, neuronal maintenance, and hematopoiesis. An obligate step in the activation of this pathway is cleavage of its transmembrane (TM) domain by γ-secretase. While the soluble domains have been extensively studied, little has been done to characterize its TM and flanking juxtamembrane (JM) segments. Here, we present the results of nuclear magnetic resonance (NMR) studies of the human Notch1 TM/JM domain. The TM domain is largely α-helical. While the flanking JM segments do not adopt regular secondary structure, they interact with the membrane surface, suggesting membrane interactions may play a role in modulating its cleavage by γ-secretase and subsequent NOTCH signaling function.


Assuntos
Bicamadas Lipídicas/química , Modelos Moleculares , Receptor Notch1/química , Humanos , Bicamadas Lipídicas/metabolismo , Lisofosfatidilcolinas/química , Lisofosfatidilcolinas/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Propriedades de Superfície
10.
Biochem Biophys Res Commun ; 459(1): 87-93, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25712527

RESUMO

Our recent study has shown that cellular junctions in myelin and in the epi-/perineruium that encase nerve fibers regulate the permeability of the peripheral nerves. This permeability may affect propagation of the action potential. Direct interactions between the PDZ1 domain of zonula occludens (ZO1 or ZO2) and the C-termini of claudins are known to be crucial for the formation of tight junctions. Using the purified PDZ1 domain of ZO2 and a variety of C-terminal mutants of peripheral nerve claudins (claudin-1, claudin-2, claudin-3, claudin-5 in epi-/perineurium; claudin-19 in myelin), we have utilized NMR spectroscopy to determine specific roles of the 3 C-terminal claudin residues (position -2, -1, 0) for their interactions with PDZ1 of ZO2. In contrast to the canonical model that emphasizes the importance of residues at the -2 and 0 positions, our results demonstrate that, for peripheral nerve claudins, the residue at position -1 plays a critical role in association with PDZ1, while the side-chain of residue 0 plays a significant but lesser role. Surprisingly, claudin-19, the most abundant claudin in myelin, exhibited no binding to ZO2. These findings reveal that the binding mechanism of claudin/ZO in epi-/perineurium is distinct from the canonical interactions between non-ZO PDZ-containing proteins with their ligands. This observation provides the molecular basis for a strategy to develop drugs that target tight junctions in the epi-/perineurium of peripheral nerves.


Assuntos
Claudinas/metabolismo , Nervos Periféricos/metabolismo , Proteína da Zônula de Oclusão-2/química , Motivos de Aminoácidos , Claudina-1/química , Claudina-1/genética , Claudina-1/metabolismo , Claudina-2/química , Claudina-2/metabolismo , Claudina-3/química , Claudina-3/metabolismo , Claudina-5/química , Claudina-5/metabolismo , Claudinas/química , Claudinas/genética , Humanos , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Proteína da Zônula de Oclusão-2/genética , Proteína da Zônula de Oclusão-2/metabolismo
11.
Cell Biosci ; 4(1): 52, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25264482

RESUMO

BACKGROUND: Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin and also the epimerization of DHNP to 7,8-dihydromonapterin. Previously, we determined the crystal structure of Staphylococcus aureus DHNA (SaDHNA) in complex with the substrate analogue neopterin (NP). We also showed that Escherichia coli DHNA (EcDHNA) and SaDHNA have significantly different binding and catalytic properties by biochemical analysis. On the basis of these structural and functional data, we proposed a catalytic mechanism involving two proton wires. RESULTS: To understand the structural basis for the biochemical differences and further investigate the catalytic mechanism of DHNA, we have determined the structure of EcDHNA complexed with NP at 1.07-Å resolution [PDB:2O90], built an atomic model of EcDHNA complexed with the substrate DHNP, and performed molecular dynamics (MD) simulation analysis of the substrate complex. EcDHNA has the same fold as SaDHNA and also forms an octamer that consists of two tetramers, but the packing of one tetramer with the other is significantly different between the two enzymes. Furthermore, the structures reveal significant differences in the vicinity of the active site, particularly in the loop that connects strands ß3 and ß4, mainly due to the substitution of nearby residues. The building of an atomic model of the complex of EcDHNA and the substrate DHNP and the MD simulation of the complex show that some of the hydrogen bonds between the substrate and the enzyme are persistent, whereas others are transient. The substrate binding model and MD simulation provide the molecular basis for the biochemical behaviors of the enzyme, including noncooperative substrate binding, indiscrimination of a pair of epimers as the substrates, proton wire switching during catalysis, and formation of epimerization product. CONCLUSIONS: The EcDHNA and SaDHNA structures, each in complex with NP, reveal the basis for the biochemical differences between EcDHNA and SaDHNA. The atomic substrate binding model and MD simulation offer insights into substrate binding and catalysis by DHNA. The EcDHNA structure also affords an opportunity to develop antimicrobials specific for Gram-negative bacteria, as DHNAs from Gram-negative bacteria are highly homologous and E. coli is a representative of this class of bacteria.

12.
J Bacteriol ; 196(12): 2131-42, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24584501

RESUMO

SpoIIID is evolutionarily conserved in endospore-forming bacteria, and it activates or represses many genes during sporulation of Bacillus subtilis. An SpoIIID monomer binds DNA with high affinity and moderate sequence specificity. In addition to a predicted helix-turn-helix motif, SpoIIID has a C-terminal basic region that contributes to DNA binding. The nuclear magnetic resonance (NMR) solution structure of SpoIIID in complex with DNA revealed that SpoIIID does indeed have a helix-turn-helix domain and that it has a novel C-terminal helical extension. Residues in both of these regions interact with DNA, based on the NMR data and on the effects on DNA binding in vitro of SpoIIID with single-alanine substitutions. These data, as well as sequence conservation in SpoIIID binding sites, were used for information-driven docking to model the SpoIIID-DNA complex. The modeling resulted in a single cluster of models in which the recognition helix of the helix-turn-helix domain interacts with the major groove of DNA, as expected. Interestingly, the C-terminal extension, which includes two helices connected by a kink, interacts with the adjacent minor groove of DNA in the models. This predicted novel mode of binding is proposed to explain how a monomer of SpoIIID achieves high-affinity DNA binding. Since SpoIIID is conserved only in endospore-forming bacteria, which include important pathogenic Bacilli and Clostridia, whose ability to sporulate contributes to their environmental persistence, the interaction of the C-terminal extension of SpoIIID with DNA is a potential target for development of sporulation inhibitors.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Fatores de Transcrição/genética
13.
J Am Chem Soc ; 136(11): 4093-6, 2014 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-24564538

RESUMO

C99 (also known as ß-CTF) is the 99 residue transmembrane C-terminal domain (residues 672-770) of the amyloid precursor protein and is the immediate precursor of the amyloid-ß (Aß) polypeptides. To test the dependence of the C99 structure on the composition of the host model membranes, NMR studies of C99 were conducted both in anionic lyso-myristoylphosphatidylglycerol (LMPG) micelles and in a series of five zwitterionic bicelle compositions involving phosphatidylcholine and sphingomyelin in which the acyl chain lengths of these lipid components varied from 14 to 24 carbons. Some of these mixtures are reported for the first time in this work and should be of broad utility in membrane protein research. The site-specific backbone (15)N and (1)H chemical shifts for C99 in LMPG and in all five bicelle mixtures were seen to be remarkably similar, indicating little dependence of the backbone structure of C99 on the composition of the host model membrane. However, the length of the transmembrane span was seen to vary in a manner that alters the positioning of the γ-secretase cleavage sites with respect to the center of the bilayer. This observation may contribute to the known dependency of the Aß42-to-Aß40 production ratio on both membrane thickness and the length of the C99 transmembrane domain.


Assuntos
Precursor de Proteína beta-Amiloide/química , Bicamadas Lipídicas/química , Fragmentos de Peptídeos/química , Humanos , Micelas , Ressonância Magnética Nuclear Biomolecular
14.
Biochemistry ; 52(8): 1303-20, 2013 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-23368985

RESUMO

From roughly 1985 through the start of the new millennium, the cutting edge of solution protein nuclear magnetic resonance (NMR) spectroscopy was to a significant extent driven by the aspiration to determine structures. Here we survey recent advances in protein NMR that herald a renaissance in which a number of its most important applications reflect the broad problem-solving capability displayed by this method during its classical era during the 1970s and early 1980s.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Animais , Descoberta de Drogas , História do Século XX , História do Século XXI , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/história , Conformação Proteica , Mapeamento de Interação de Proteínas/história , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo
15.
Mol Cell Biol ; 32(20): 4080-91, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22869523

RESUMO

Loss of ß1 integrin expression inhibits renal collecting-system development. Two highly conserved NPXY motifs in the distal ß1 tail regulate integrin function by associating with phosphtyrosine binding (PTB) proteins, such as talin and kindlin. Here, we define the roles of these two tyrosines in collecting-system development and delineate the structural determinants of the distal ß1 tail using nuclear magnetic resonance (NMR). Mice carrying alanine mutations have moderate renal collecting-system developmental abnormalities relative to ß1-null mice. Phenylalanine mutations did not affect renal collecting-system development but increased susceptibility to renal injury. NMR spectra in bicelles showed the distal ß1 tail is disordered and does not interact with the model membrane surface. Alanine or phenylalanine mutations did not alter ß1 structure or interactions between α and ß1 subunit transmembrane/cytoplasmic domains; however, they did decrease talin and kindlin binding. Thus, these studies highlight the fact that the functional roles of the NPXY motifs are organ dependent. Moreover, the ß1 cytoplasmic tail, in the context of the adjacent transmembrane domain in bicelles, is significantly different from the more ordered, membrane-associated ß3 integrin tail. Finally, tyrosine mutations of ß1 NPXY motifs induce phenotypes by disrupting their interactions with critical integrin binding proteins like talins and kindlins.


Assuntos
Integrina beta1/metabolismo , Túbulos Renais Coletores/crescimento & desenvolvimento , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Citosol/metabolismo , Humanos , Integrina beta1/genética , Integrina beta3/química , Integrina beta3/metabolismo , Proteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Mutação , Proteínas de Neoplasias/química , Ligação Proteica , Conformação Proteica , Talina/química , Tirosina/química , Tirosina/genética , Tirosina/metabolismo
16.
Mol Pharm ; 9(4): 752-61, 2012 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-22221179

RESUMO

Bilayered detergent-lipid assemblies known as bicelles have been widely used as model membranes in structural biological studies and are being explored for wider applications, including pharmaceutical use. Most studies to date have involved the use of concentrated bicelle mixtures, such that little is known about the capacity of bicellar mixtures to be diluted without unwanted transitions to nonisotropic phases. Here, different detergent/lipid mixtures have been explored, leading to the identification of two different families of bicelles for which it is possible to lower the total amphiphile (detergent + lipid) concentration to <1% (w/v) while retaining isotropic assemblies. These include a novel family of bicelles based on mixtures of 6-cyclohexyl-1-hexylphosphocholine (Cyclofos-6) and the lipid dimyristoylphosphatidylcholine (DMPC). Bicelles formed by these mixtures can be diluted to <0.5% and also have attractive biochemical properties. However, a caveat of our results is that the diffusion coefficients measured for the lipid component of the different bicelles tested were seen to be dependent on sample history, even though all samples were optically transparent. This suggests that the phase behavior of bicelles at low lipid-to-detergent ratios may be more complex than previously appreciated.


Assuntos
Detergentes/química , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Micelas
17.
Biochemistry ; 48(2): 302-12, 2009 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-19108643

RESUMO

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), which follows an ordered bi-bi kinetic mechanism with ATP binding to the enzyme first. HPPK undergoes dramatic conformational changes during its catalytic cycle as revealed by X-ray crystallography, and the conformational changes are essential for the enzymatic catalysis as shown by site-directed mutagenesis and biochemical and crystallographic analysis of the mutants. However, the dynamic properties of the enzyme have not been measured experimentally. Here, we report a (15)N NMR relaxation study of the dynamic properties of Escherichia coli HPPK from the apo form to the binary substrate complex with MgATP (represented by MgAMPCPP, an ATP analogue) to the Michaelis complex (ternary substrate complex) with MgATP (represented by MgAMPCPP) and HP (represented by 7,7-dimethyl-6-hydroxypterin, an HP analogue). The results show that the binding of the nucleotide to HPPK does not cause major changes in the dynamic properties of the enzyme. Whereas enzymes are often more rigid when bound to the ligand or the substrate, the internal mobility of HPPK is not reduced and is even moderately increased in the binary complex, particularly in the catalytic loops. The internal mobility of the catalytic loops is significantly quenched upon the formation of the ternary complex, but some mobility remains. The enhanced motions in the catalytic loops of the binary substrate complex may be required for the assembling of the ternary complex. On the other hand, some degrees of mobility in the catalytic loops of the ternary complex may be required for the optimal stabilization of the transition state, which may need the instantaneous adjustment and alignment of the side-chain positions of catalytic residues. Such dynamic behaviors may be characteristic of bisubstrate enzymes.


Assuntos
Difosfotransferases/metabolismo , Escherichia coli/enzimologia , Conformação Proteica , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Difusão , Difosfotransferases/química , Difosfotransferases/isolamento & purificação , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Rotação , Especificidade por Substrato/genética , Temperatura Ambiente
18.
Chembiochem ; 9(17): 2860-71, 2008 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-18973166

RESUMO

The configuration and hydrogen-bonding network of side-chain amides in a 35 kDa protein were determined by measuring differential and trans-hydrogen-bond H/D isotope effects by using the isotopomer-selective (IS)-TROSY technique, which leads to a reliable recognition and correction of erroneous rotamers that are frequently found in protein structures. First, the differential two-bond isotope effects on carbonyl (13)C' shifts, which are defined as Delta(2)Delta(13)C'(ND) = (2)Delta(13)C'(ND(E))-(2)Delta(13)C'(ND(Z)), provide a reliable means for the configuration assignment for side-chain amides, because environmental effects (hydrogen bonds and charges, etc.) are greatly attenuated over the two bonds that separate the carbon and hydrogen atoms, and the isotope effects fall into a narrow range of positive values. Second and more importantly, the significant variations in the differential one-bond isotope effects on (15)N chemical shifts, which are defined as Delta(1)Delta(15)N(D) = (1)Delta(15)N(D(E))-(1)Delta(15)N(D(Z)) can be correlated with hydrogen-bonding interactions, particularly those involving charged acceptors. The differential one-bond isotope effects are additive, with major contributions from intrinsic differential conjugative interactions between the E and Z configurations, H-bonding interactions, and charge effects. Furthermore, the pattern of trans-H-bond H/D isotope effects can be mapped onto more complicated hydrogen-bonding networks that involve bifurcated hydrogen-bonds. Third, the correlations between Delta(1)Delta(15)N(D) and hydrogen-bonding interactions afford an effective means for the correction of erroneous rotamer assignments of side-chain amides. Rotamer correction by differential isotope effects is not only robust, but also simple and can be applied to large proteins.


Assuntos
Amidas/química , Citosina Desaminase/química , Ressonância Magnética Nuclear Biomolecular , Deutério , Ligações de Hidrogênio , Marcação por Isótopo , Isótopos de Nitrogênio , Conformação Proteica , Prótons , Leveduras/enzimologia
20.
J Appl Physiol (1985) ; 101(2): 598-608, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16849813

RESUMO

We present an evaluation of a novel technique for continuous (i.e., automatic) monitoring of relative cardiac output (CO) changes by long time interval analysis of a peripheral arterial blood pressure (ABP) waveform in humans. We specifically tested the mathematical analysis technique based on existing invasive and noninvasive hemodynamic data sets. With the former data set, we compared the application of the technique to peripheral ABP waveforms obtained via radial artery catheterization with simultaneous thermodilution CO measurements in 15 intensive care unit patients in which CO was changing because of disease progression and therapy. With the latter data set, we compared the application of the technique to noninvasive peripheral ABP waveforms obtained via a finger-cuff photoplethysmography system with simultaneous Doppler ultrasound CO measurements made by an expert in 10 healthy subjects during pharmacological and postural interventions. We report an overall CO root-mean-squared normalized error of 15.3% with respect to the invasive hemodynamic data set and 15.1% with respect to the noninvasive hemodynamic data set. Moreover, the CO errors from the invasive and noninvasive hemodynamic data sets were only mildly correlated with mean ABP (rho = 0.41, 0.37) and even less correlated with CO (rho = -0.14, -0.17), heart rate (rho = 0.04, 0.19), total peripheral resistance (rho = 0.38, 0.10), CO changes (rho = -0.26, -0.20), and absolute CO changes (rho = 0.03, 0.38). With further development and successful prospective testing, the technique may potentially be employed for continuous hemodynamic monitoring in the acute setting such as critical care and emergency care.


Assuntos
Monitorização Ambulatorial da Pressão Arterial/métodos , Pressão Sanguínea/fisiologia , Débito Cardíaco/fisiologia , Monitorização Fisiológica/métodos , Idoso , Algoritmos , Monóxido de Carbono/análise , Ecocardiografia Doppler/métodos , Feminino , Hemodinâmica/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Teóricos , Fotopletismografia/métodos , Volume Sistólico/fisiologia , Fatores de Tempo
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