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101.
Katsamba PS Bayramyan M Haworth IS Myszka DG Laird-Offringa IA 《The Journal of biological chemistry》2002,277(36):33267-33274
RNA recognition motifs (RRMs) are characterized by highly conserved regions located centrally on a beta-sheet, which forms the RNA binding surface. Variable flanking regions, such as the loop connecting beta-strands 2 and 3, are thought to be important in determining the RNA-binding specificities of individual RRMs. The N-terminal RRM of the spliceosomal U1A protein mediates binding to an RNA hairpin (U1hpII) in the U1 small nuclear RNA. In this complex, the beta(2)-beta(3) loop protrudes through the 10-nucleotide RNA loop. Shortening of the RNA loop strongly perturbs binding, suggesting that an optimal "fit" of the beta(2)-beta(3) loop into the RNA loop is an important factor in complexation. To understand this interaction further, we mutated or deleted loop residues Lys(50) and Met(51), which protrude centrally into the RNA loop but do not make any direct contacts to the bases. Using BIACORE, we analyzed the ability of these U1A mutants to bind to wild type RNAs, or RNAs with shortened loops. Alanine replacement mutations only modestly affected binding to wild type U1hpII. Interestingly, simultaneous replacement of Lys(50) and Met(51) with alanine appeared to alleviate the loss of binding caused by shortening of the RNA loop. Deletion of Lys(50) or Met(51) caused a dramatic loss in stability of the U1A.U1hpII complex. However, deletion of both residues simultaneously was much less deleterious. Simulated annealing molecular dynamics analyses suggest this is due to the ability of this mutant to rearrange flanking amino acids to substitute for the two deleted residues. The double deletion mutant also exhibited substantially reduced negative effects of RNA loop shortening, suggesting the rearranged loop is better able to accommodate a short RNA loop. Our results indicate that one of the roles of the beta(2)-beta(3) loop is to provide a steric fit into the RNA loop, thereby stabilizing the RNA.protein complex. 相似文献
102.
Crystal structure of the PH-BEACH domains of human LRBA/BGL 总被引:1,自引:0,他引:1
The beige and Chediak-Higashi syndrome (BEACH) domain defines a large family of eukaryotic proteins that have diverse cellular functions in vesicle trafficking, membrane dynamics, and receptor signaling. The domain is the only module that is highly conserved among all of these proteins, but the exact functions of this domain and the molecular basis for its actions are currently unknown. Our previous studies showed that the BEACH domain is preceded by a novel, weakly conserved pleckstrin homology (PH) domain. We report here the crystal structure at 2.4 A resolution of the PH-BEACH domain of human LRBA/BGL. The PH domain has the same backbone fold as canonical PH domains, despite sharing no sequence homology with them. However, our binding assays demonstrate that the PH domain in the BEACH proteins cannot bind phospholipids. The BEACH domain contains a core of several partially extended peptide segments that is flanked by helices on both sides. The structure suggests intimate association between the PH and the BEACH domains, and surface plasmon resonance studies confirm that the two domains of the protein FAN have high affinity for each other, with a K(d) of 120 nM. 相似文献
103.
Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods 总被引:2,自引:0,他引:2
Two biophysical methods, Biacore and KinExA, were used to kinetically and thermodynamically characterize high-affinity antigen/antibody complexes. Three to five independent experiments were performed on each platform with three different antigen/antibody complexes possessing nanomolar to picomolar equilibrium dissociation constants. By monitoring the dissociation phase on Biacore for 4 h, we were able to measure dissociation rate constants (kd) on the order of 1 x 10(-5)s(-1). To characterize high-affinity interactions by KinExA, samples needed to be equilibrated for up to 35 h to reach equilibrium. In the end, we show that similar kinetic rate constants and affinities were determined with both solution-phase and solid-phase methodologies. These results help further validate both interaction technologies and illustrate their suitability for characterizing extremely high-affinity interactions. 相似文献
104.
Survey of the year 2001 commercial optical biosensor literature 总被引:3,自引:0,他引:3
We have assembled references of 700 articles published in 2001 that describe work performed using commercially available optical biosensors. To illustrate the technology's diversity, the citation list is divided into reviews, methods and specific applications, as well as instrument type. We noted marked improvements in the utilization of biosensors and the presentation of kinetic data over previous years. These advances reflect a maturing of the technology, which has become a standard method for characterizing biomolecular interactions. 相似文献
105.
106.
The Ser/Thr-specific IkappaB kinase (IKK), which comprises IKKalpha or IKKbeta and the regulatory protein NEMO, is at the bottleneck for NF-kappaB activation. IKK activity relies on interaction between NEMO and IKKalpha or IKKbeta. A conserved region in the C-terminal tail of IKKbeta or IKKalpha (NEMO-binding domain, NBD, residues 734-745 of IKKbeta) is important for interaction with NEMO. Here we show that the NBD peptide of IKKbeta is not sufficient for interaction with NEMO. Instead, a longer region of the IKKbeta C-terminal region provides high affinity for NEMO. Quantitative measurements using surface plasmon resonance and isothermal titration calorimetry confirm the differential affinities of these interactions and provide insight into the kinetic and thermodynamic behaviors of the interactions. Biochemical characterization using multiangle light scattering (MALS) coupled with refractive index shows that the longer IKKbeta C-terminal region forms a 2:2 stoichiometirc complex with NEMO. 相似文献
107.
The classical method of measuring binding constants with affinity-based biosensors involves testing several analyte concentrations over the same ligand surface and regenerating the surface between binding cycles. Here we describe an alternative approach to collecting kinetic binding data, which we call "kinetic titration." This method involves sequentially injecting an analyte concentration series without any regeneration steps. Through a combination of simulation and experimentation, we show that this method can be as robust as the classical method of analysis. In addition, kinetic titrations can be more efficient than the conventional data collection method and allow us to fully characterize analyte binding to ligand surfaces that are difficult to regenerate. 相似文献
108.
Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users 总被引:1,自引:0,他引:1
Katsamba PS Navratilova I Calderon-Cacia M Fan L Thornton K Zhu M Bos TV Forte C Friend D Laird-Offringa I Tavares G Whatley J Shi E Widom A Lindquist KC Klakamp S Drake A Bohmann D Roell M Rose L Dorocke J Roth B Luginbühl B Myszka DG 《Analytical biochemistry》2006,352(2):208-221
To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5-600 nM to obtain k(a) information. Second, to define the k(d) of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average k(a) and k(d) for the PSA/mAb interaction as calculated from the 22 analyses were (4.1+/-0.6) x 10(4) M(-1) s(-1) and (4.5+/-0.6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM. 相似文献
109.
Myszka A Karpinski P Slezak R Czemarmazowicz H Stembalska A Gil J Laczmanska I Bednarczyk D Szmida E Sasiadek MM 《Journal of applied genetics》2011,52(2):185-191
CHEK2 gen encodes cell cycle checkpoint kinase 2 that participates in the DNA repair pathway, cell cycle regulation and apoptosis.
Mutations in CHEK2 gene may result in kinase inactivation or reduce both catalytic activity and capability of binding other proteins. Some studies
indicate that alterations in CHEK2 gene confers increase the risk of breast cancer and some other malignancies, while the results of other studies are inconclusive.
Thus the significance of CHEK2 mutations in aetiology of breast cancer is still debatable. The aim of our study was to evaluate the relationship between
the breast/ovarian cancer and CHEK2 variants by: i) the analysis of the frequency of selected CHEK2 variants in breast and ovarian cancer patients compared to the controls; ii) evaluation of relationships between the certain CHEK2 variants and clinico-histopathological and pedigree data. The study was performed on 284 breast cancer patients, 113 ovarian
cancer patients and 287 healthy women. We revealed the presence of 430T > C, del5395 and IVS2 + 1G > A variants but not 1100delC
in individuals from both study and control groups. We did not observe significant differences between cancer patients and
controls neither in regard to the frequency nor to the type of CHEK2 variants. We discussed the potential application of CHEK2 variants in the evaluation of breast and ovarian cancer predisposition. 相似文献
110.
Fabíola C Toledo Juliana E Perobelli Flávia PC Pedrosa Janete A Anselmo-Franci Wilma DG Kempinas 《Reproductive biology and endocrinology : RB&E》2011,9(1):1-9