共查询到20条相似文献,搜索用时 15 毫秒
1.
Turner Sarah L.; Russell George C.; Williamson Michael P.; Guest John R. 《Protein engineering, design & selection : PEDS》1993,6(1):101-108
The lipoyl, subunit-binding and catalytic domains of the dihydrolipoamideacetyltransferase subunits (E2p) of the Escherichia coli pyruvatedehydrogenase complex are connected by linker sequences whichare characteristically rich in alanine and proline residues.By facilitating domain movement these linkers are thought topromote interactions between the three types of active sitethat participate in the catalytic cycle of the complex. To investigatefunctional constraints associated with linker composition andsequence, the natural linker of an E2p subunit containing onelipoyl domain was replaced by shorter sequences containing:mixtures of alanine plus proline residues; mainly alanine; mainlyproline; and mainly charged residues. Each artificial linkerpossessed a central histidine residue for assessing linker flexibilityby 1H-NMR spectroscopy. The resultant complexes exhibited 181%(proline), 7479% (alanine plus proline), 63% (alanine)and 7% (charged residues) of parental activity compared witha value of 75% expected for a complex with a comparably shortenedlinker. The 1H-NMR spectra showed that the alanine plus prolinelinkers are flexible but the alanine linker and the prolinelinker are relatively inflexible. Substantial variations inlinker sequence and composition were tolerated without lossof function, and the enhanced activity conferred by the prolinelinker was attributed to the combined effects of length andrelative inflexibility. 相似文献
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3.
Keiichi Asano Anna Cantalupo Lauriane Sedes Francesco Ramirez 《International journal of molecular sciences》2022,23(3)
Fibrillin-1 is the major structural component of the 10 nm-diameter microfibrils that confer key physical and mechanical properties to virtually every tissue, alone and together with elastin in the elastic fibers. Mutations in fibrillin-1 cause pleiotropic manifestations in Marfan syndrome (MFS), including dissecting thoracic aortic aneurysms, myocardial dysfunction, progressive bone loss, disproportionate skeletal growth, and the dislocation of the crystalline lens. The characterization of these MFS manifestations in mice, that replicate the human phenotype, have revealed that the underlying mechanisms are distinct and organ-specific. This brief review summarizes relevant findings supporting this conclusion. 相似文献
4.
Langer T Vogtherr M Elshorst B Betz M Schieborr U Saxena K Schwalbe H 《Chembiochem : a European journal of chemical biology》2004,5(11):1508-1516
Protein phosphorylation is one of the most important mechanisms used for intracellular regulation in eukaryotic cells. Currently, one of the best-characterized protein kinases is the catalytic subunit of cAMP-dependent protein kinase or protein kinase A (PKA). PKA has the typical bilobular structure of kinases, with the active site consisting of a cleft between the two structural lobes. For full kinase activity, the catalytic subunit has to be phosphorylated. The catalytic subunit of PKA has two main phosphorylation sites: Thr197 and Ser338. Binding of ATP or inhibitors to the ATP site induces large structural changes. Here we describe the partial backbone assignment of the PKA catalytic domain by NMR spectroscopy, which represents the first NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment for the 42 kDa protein was accomplished by an approach employing 1) triply ((2)H,(13)C,(15)N) labeled protein and classical NMR assignment experiments, 2) back-calculation of chemical shifts from known X-ray structures, 3) use of paramagnetic adenosine derivatives as spin-labels, and 4) selective amino acid labeling. Interpretation of chemical-shift perturbations allowed mapping of the interaction surface with the protein kinase inhibitor H7. Furthermore, structural conformational changes were observed by comparison of backbone amide shifts obtained by 2D (1)H,(15)N TROSY of an inactive Thr197Ala mutant with the wild-type enzyme. 相似文献
5.
Liang Tiebing; Chen Jinqiu; Tjornhammar Marie-Louise; Pongor Sandor; Simoncsits Andras 《Protein engineering, design & selection : PEDS》2001,14(8):591-599
Single-chain derivatives of the 434 repressor containing onewild-type and one mutant DNA-binding domain recognize the generaloperator ACAA6 base pairsNNNN, where the ACAAoperator subsite is contacted by the wild-type and the NNNNtetramer by the mutant domain. The DNA-binding specificitiesof several single-chain mutants were studied in detail and theoptimal subsites of the mutant domains were determined. Thecharacterized mutant domains were used as building units toobtain homo- and heterodimeric single-chain derivatives. TheDNA-binding properties of these domain-shuffled derivativeswere tested with a series of designed operators of NNNN6base pairsNNNN type. It was found that the binding specificitiesof the mutant domains were generally maintained in the new environmentsand the binding affinities for the optimal DNA ligands werehigh (with Kd values in the range of 10111010M). Considering that only certain sequence motifs in place ofthe six base pair spacer can support optimal contacts betweenthe mutant domains and their subsites, the single-chain 434repressor mutants are highly specific for a limited subset of14 base pair long DNA targets. 相似文献
6.
The pedicel, nest paper, and larval silk ofPolistes annularis nests were analyzed by high-resolution solid-state [13C]NMR. The pedicel was found to have a high nitrogen content (11%), and the NMR spectra indicated that it is a mixture of carbohydrate and protein. The pedicel protein has an amino acid composition that is very rich in glycine, alanine, serine, and proline (67% of identified residues), similar to that of some insect silks. Solid-state [13C]NMR indicated that the nest paper is composed predominantly of cellulose. Silk, spun by matureP. annularis larvae, was shown by [13C] NMR and amino acid analysis to be a protein very high in serine and alanine (53%), but the amino acid composition is distinct from that of the pedicel protein. 相似文献
7.
Theresa L. Singer Karl E. Espelie David S. Himmelsbach 《Journal of chemical ecology》1992,18(1):77-86
The ultrastructure and chemical composition of paper and pedicel from nests ofPolistes metricus that were constructed in the laboratory from known building material were compared to paper and pedicel from nests constructed in the field. Scanning electron micrographs showed the addition of a gluelike secretion from the wasp to the construction material. Solid-state [13C]NMR, elemental analyses, and amino acid analyses indicate that this secretion is a silklike protein with serine, glycine, alanine, and proline comprising 65–73% of the identified residues. 相似文献
8.
Qu C; Akanuma S; Moriyama H; Tanaka N; Oshima T 《Protein engineering, design & selection : PEDS》1997,10(1):45-52
The structure of a thermostable Ala172Leu mutant, designated A172L, of
3-isopropylmalate dehydrogenase from Thermus thermophilus was determined.
The crystal belongs to space group P2(1), with cell parameters a = 55.5 A,
b = 88.1 A, c = 72.0 A and beta = 100.9 degrees. There is one dimer in each
asymmetric unit. The final R factor is 17.8% with 69 water molecules at
2.35 A resolution. The mutation is located at the interface between domains
and the C alpha trace of the mutant structure deviates from that of the
native structure by as much as 1.7 A, while the structure of each domain
barely changes. The mutant enzyme has a more closed conformation compared
with the wild-type enzyme as a result of the replacement of Ala with Leu at
residue 172. These structural variations were found independent of the
crystal packing, because the structure of wild type was the same in
crystals obtained in different precipitants. The hinge regions for the
movement of domains are located around the active cleft of the enzyme, an
observation that implies that the mobility of domains around the hinge is
indispensable for the activity of the enzyme. The larger side chain at the
mutated site contributed to the thermostability of the mutant protein by
enhancing the local packing of side chains, and also by shifting the
backbone of the opposing domain.
相似文献
9.
Manisha Kumari Shahu Fabian Schuhmann Alexander Scholten Ilia A. Solovyov Karl-Wilhelm Koch 《International journal of molecular sciences》2022,23(7)
Membrane-bound guanylate cyclases (GCs), which synthesize the second messenger guanosine-3′, 5′-cyclic monophosphate, differ in their activation modes to reach the active state. Hormone peptides bind to the extracellular domain in hormone-receptor-type GCs and trigger a conformational change in the intracellular, cytoplasmic part of the enzyme. Sensory GCs that are present in rod and cone photoreceptor cells have intracellular binding sites for regulatory Ca2+-sensor proteins, named guanylate-cyclase-activating proteins. A rotation model of activation involving an α-helix rotation was described as a common activation motif among hormone-receptor GCs. We tested whether the photoreceptor GC-E underwent an α-helix rotation when reaching the active state. We experimentally simulated such a transitory switch by integrating alanine residues close to the transmembrane region, and compared the effects of alanine integration with the point mutation V902L in GC-E. The V902L mutation is found in patients suffering from retinal cone–rod dystrophies, and leads to a constitutively active state of GC-E. We analyzed the enzymatic catalytic parameters of wild-type and mutant GC-E. Our data showed no involvement of an α-helix rotation when reaching the active state, indicating a difference in hormone receptor GCs. To characterize the protein conformations that represent the transition to the active state, we investigated the protein dynamics by using a computational approach based on all-atom molecular dynamics simulations. We detected a swinging movement of the dimerization domain in the V902L mutant as the critical conformational switch in the cyclase going from the low to high activity state. 相似文献
10.
Phonghanpot S Punya J Tachaleat A Laoteng K Bhavakul V Tanticharoen M Cheevadhanarak S 《Chembiochem : a European journal of chemical biology》2012,13(6):895-903
A gene from Xylaria sp. BCC 1067, pks3, that encodes a putative 3660-residue hybrid polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) was characterised by targeted gene disruption in combination with comprehensive product identification. Studies of the features of a corresponding mutant, YA3, allowed us to demonstrate that pks3 is responsible for the synthesis of a new pyrroline compound, named xyrrolin, in the wild-type Xylaria sp. BCC 1067. The structure of xyrrolin was established by extensive spectroscopic and spectrometric analyses, including low- and high-resolution MS, IR, (1)H NMR, (13)C NMR, (13)C NMR with Dept135, HMQC 2D NMR, HMBC 2D NMR and COSY 2D NMR. On the basis of the Pks3 domain organisation and the chemical structure of xyrrolin, we proposed that biosynthesis of this compound requires the condensation of a tetraketide and an L-serine unit, followed by Dieckmann or reductive cyclisation and enzymatic removal of ketone residue(s). Bioassays of the pure xyrrolin further displayed cytotoxicity against an oral cavity (KB) cancer cell line. 相似文献
11.
Cytochrome P450cam dimerizes via the formation of an intermolecular
disulfide bond, complicating the storage and handling of the enzyme,
particularly at higher concentrations. The dimeric enzyme is 14% less
active than the monomer and forms at a slow but significant rate even at 4
degrees C [k = 1.09 x 10(-3) mM(-1) h(-1)]. To eliminate any ambiguity
introduced by dimer formation and to simplify handling and storage of the
enzyme, site-directed mutagenesis was used to identify C334 as the single
cysteine residue responsible for the formation of the disulfide linkage and
to engineer a monomeric enzyme by substituting an alanine in its place. The
C334A mutant is identical with the wild-type P450cam monomer in terms of
optical spectra, camphor binding and turnover activity, but shows no
evidence of dimerization and aggregation even at millimolar concentrations.
Preliminary 1H NMR investigations also indicate a significant improvement
in the quality of spectra obtained with this mutant. (C334A)P450cam is
therefore proposed as an alternative to the wild-type enzyme-a base mutant
otherwise identical with the wild-type but with improved handling
characteristics.
相似文献
12.
Mutant huntingtin (m-HTT) proteins and calmodulin (CaM) co-localize in the cerebral cortex with significant effects on the intracellular calcium levels by altering the specific calcium-mediated signals. Furthermore, the mutant huntingtin proteins show great affinity for CaM that can lead to a further stabilization of the mutant huntingtin aggregates. In this context, the present study focuses on describing the interactions between CaM and two huntingtin mutants from a biophysical point of view, by using classical Molecular Dynamics techniques. The huntingtin models consist of a wild-type structure, one mutant with 45 glutamine residues and the second mutant with nine additional key-point mutations from glutamine residues into proline residues (9P(EM) model). Our docking scores and binding free energy calculations show higher binding affinities of all HTT models for the C-lobe end of the CaM protein. In terms of dynamic evolution, the 9P(EM) model triggered great structural changes into the CaM protein’s structure and shows the highest fluctuation rates due to its structural transitions at the helical level from α-helices to turns and random coils. Moreover, our proposed 9P(EM) model suggests much lower interaction energies when compared to the 45Qs-HTT mutant model, this finding being in good agreement with the 9P(EM)’s antagonistic effect hypothesis on highly toxic protein–protein interactions. 相似文献
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14.
Dr. Abhishek Cukkemane Prof. Dr. Marc Baldus 《Chembiochem : a European journal of chemical biology》2013,14(14):1789-1798
Voltage‐gated ion channels are large tetrameric multidomain membrane proteins that play crucial roles in various cellular transduction pathways. Because of their large size and domain‐related mobility, structural characterization has proved challenging. We analyzed high‐resolution solid‐state NMR data on different isotope‐labeled protein constructs of a bacterial cyclic nucleotide‐activated K+ channel (MlCNG) in lipid bilayers. We could identify the different subdomains of the 4×355 residue protein, such as the voltage‐sensing domain and the cyclic nucleotide binding domain. Comparison to ssNMR data obtained on isotope‐labeled cell membranes suggests a tight association of negatively charged lipids to the channel. We detected spectroscopic polymorphism that extends beyond the ligand binding site, and the corresponding protein segments have been associated with mutant channel types in eukaryotic systems. These findings illustrate the potential of ssNMR for structural investigations on large membrane‐embedded proteins, even in the presence of local disorder. 相似文献
15.
Yumiko Tahira Katsuya Sakai Hiroki Sato Ryu Imamura Kunio Matsumoto 《International journal of molecular sciences》2021,22(17)
NK1, a splicing variant of hepatocyte growth factor (HGF), binds to and activates Met receptor by forming an NK1 dimer and 2:2 complex with Met. Although the structural mechanism underlying Met activation by HGF remains incompletely resolved, it has been proposed that the NK1 dimer structure participates in this activation. We investigated the NK1 dimer interface’s role in Met activation by HGF. Because N127, V140, and K144 are closely involved in the head-to-tail NK1 dimer formation, mutant NK1 proteins with replacement of these residues by alanine were prepared. In Met tyrosine phosphorylation assays, N127-NK1, V140-NK1, and K144-NK1 showed 8.3%, 23.8%, and 52.2% activity, respectively, compared with wild-type NK1. Although wild-type NK1 promoted cell migration and scattering, N127-NK1, V140-NK1, and K144-NK1 hardly or marginally promoted them, indicating loss of activity of these mutant NK1 proteins to activate Met. In contrast, mutant HGFs (N127-HGF, V140-HGF, and K144-HGF) with the same amino acid replacements as in NK1 induced Met tyrosine phosphorylation and biological responses at levels comparable to those of wild-type HGF. These results indicate that the structural basis responsible for NK1-dependent Met dimer formation and activation differs from, or is at least distinguishable from, the structural basis responsible for HGF-dependent Met activation. 相似文献
16.
17.
Alexander V. Fonin Sergey A. Silonov Anna S. Fefilova Olesya V. Stepanenko Anastasia A. Gavrilova Alexey V. Petukhov Anna E. Romanovich Anna L. Modina Tatiana S. Zueva Evgeniy M. Nedelyaev Nadejda M. Pleskach Mirya L. Kuranova Irina M. Kuznetsova Vladimir N. Uversky Konstantin K. Turoverov 《International journal of molecular sciences》2022,23(3)
In this work, we performed a comparative study of the formation of PML bodies by full-length PML isoforms and their C-terminal domains in the presence and absence of endogenous PML. Based on the analysis of the distribution of intrinsic disorder predisposition in the amino acid sequences of PML isoforms, regions starting from the amino acid residue 395 (i.e., sequences encoded by exons 4–6) were assigned as the C-terminal domains of these proteins. We demonstrate that each of the full-sized nuclear isoforms of PML is capable of forming nuclear liquid-droplet compartments in the absence of other PML isoforms. These droplets possess dynamic characteristics of the exchange with the nucleoplasm close to those observed in the wild-type cells. Only the C-terminal domains of the PML-II and PML-V isoforms are able to be included in the composition of the endogenous PML bodies, while being partially distributed in the nucleoplasm. The bodies formed by the C-terminal domain of the PML-II isoform are dynamic liquid droplet compartments, regardless of the presence or absence of endogenous PML. The C-terminal domain of PML-V forms dynamic liquid droplet compartments in the knockout cells (PML−/−), but when the C-terminus of the PML-V isoform is inserted into the existing endogenous PML bodies, the molecules of this protein cease to exchange with the nucleoplasm. It was demonstrated that the K490R substitution, which disrupts the PML sumoylation, promotes diffuse distribution of the C-terminal domains of PML-II and PML-V isoforms in endogenous PML knockout HeLa cells, but not in the wild-type cells. These data indicate the ability of the C-terminal domains of the PML-II and PML-V isoforms to form dynamic liquid droplet-like compartments, regardless of the ordered N-terminal RBCC motifs of the PML. This indicates a significant role of the non-specific interactions between the mostly disordered C-terminal domains of PML isoforms for the initiation of liquid–liquid phase separation (LLPS) leading to the formation of PML bodies. 相似文献
18.
Yoon-Jeong Choi Yujin Lee Yuxi Lin Yunseok Heo Young-Ho Lee Kiwon Song 《International journal of molecular sciences》2022,23(13)
The condensation of nuclear promyelocytic leukemia bodies, cytoplasmic P-granules, P-bodies (PBs), and stress granules is reversible and dynamic via liquid–liquid phase separation. Although each condensate comprises hundreds of proteins with promiscuous interactions, a few key scaffold proteins are required. Essential scaffold domain sequence elements, such as poly-Q, low-complexity regions, oligomerizing domains, and RNA-binding domains, have been evaluated to understand their roles in biomolecular condensation processes. However, the underlying mechanisms remain unclear. We analyzed Nst1, a PB-associated protein that can intrinsically induce PB component condensations when overexpressed. Various Nst1 domain deletion mutants with unique sequence distributions, including intrinsically disordered regions (IDRs) and aggregation-prone regions, were constructed based on structural predictions. The overexpression of Nst1 deletion mutants lacking the aggregation-prone domain (APD) significantly inhibited self-condensation, implicating APD as an oligomerizing domain promoting self-condensation. Remarkably, cells overexpressing the Nst1 deletion mutant of the polyampholyte domain (PD) in the IDR region (Nst1∆PD) rarely accumulate endogenous enhanced green fluorescent protein (EGFP)-tagged Dcp2. However, Nst1∆PD formed self-condensates, suggesting that Nst1 requires PD to interact with Dcp2, regardless of its self-condensation. In Nst1∆PD-overexpressing cells treated with cycloheximide (CHX), Dcp2, Xrn1, Dhh1, and Edc3 had significantly diminished condensation compared to those in CHX-treated Nst1-overexpressing cells. These observations suggest that the PD of the IDR in Nst1 functions as a hub domain interacting with other PB components. 相似文献
19.
Caffrey MS; Gooley PR; Zhao D; Meyer TE; Cusanovich MA; MacKenzie NE 《Protein engineering, design & selection : PEDS》1997,10(1):77-80
It was shown by Koshy et al. [1990, Proc. Natl Acad. Sci USA, 87, 8697-
8701; 1994, Biochem. J., 299, 347-350] that the substitution of proline 30
by alanine (P30A) of Drosophila melanogaster and rat cytochromes c
exhibited decreased stabilities in both the heme iron-methionine sulfur
(Fe-S) bond and overall protein conformation. Now we have found that the
stability properties of the equivalent mutant of Rhodobacter capsulatus
cytochrome c2 (P35A) are somewhat different. Based on optical and NMR
spectroscopies, the Rb.capsulatus P35A alkaline transition (pKalk) was
found to be unchanged with respect to the wild type, suggesting that the
mutation in Rb.capsulatus cytochrome c2 has little effect on the stability
of the Fe-S bond. However, Rb.capsulatus conformational stability was found
to be decreased by 1.6 kcal/mol in the oxidized state. The difference in
the stability properties of the equivalent proline to alanine substitutions
in various species underscores the importance of studying mutations in more
than one species before drawing generalizations about the role of conserved
residues in protein structure and function.
相似文献