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641.
To facilitate biosensor studies of G-protein coupled receptors (GPCR) and other membrane proteins, reliable methods for preparation of sensor surfaces with high protein density are required. We present here a method for the easy and rapid immobilization and reconstitution of GPCR on carboxylated dextran surfaces modified with long alkyl groups. Following amine coupling of the detergent-solubilized receptor, lipid/detergent-mixed micelles were adhered as they were injected over the immobilized surface, taking advantage of the integrated flow cells. The detergent was eluted in the subsequent buffer flow and the remaining lipid formed a bilayer on the chip surface. With this procedure, rhodopsin was functionally reconstituted in a lipid environment in approximately 1 min. This method can also be used for the easy formation of pure supported lipid bilayers for use in model membrane interaction studies. 相似文献
642.
《Journal of molecular biology》2023,435(15):168187
The strength of binding between human angiotensin converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of viral spike protein plays a role in the transmissibility of the SARS-CoV-2 virus. In this study we focus on a subset of RBD mutations that have been frequently observed in infected individuals and probe binding affinity changes to ACE2 using surface plasmon resonance (SPR) measurements and free energy perturbation (FEP) calculations. Our SPR results are largely in accord with previous studies but discrepancies do arise due to differences in experimental methods and to protocol differences even when a single method is used. Overall, we find that FEP performance is superior to that of other computational approaches examined as determined by agreement with experiment and, in particular, by its ability to identify stabilizing mutations. Moreover, the calculations successfully predict the observed cooperative stabilization of binding by the Q498R N501Y double mutant present in Omicron variants and offer a physical explanation for the underlying mechanism. Overall, our results suggest that despite the significant computational cost, FEP calculations may offer an effective strategy to understand the effects of interfacial mutations on protein–protein binding affinities and, hence, in a variety of practical applications such as the optimization of neutralizing antibodies. 相似文献
643.
Sang Yoon Hwang Chang Hoon Yoon Jun Yeoung Jeon Sung Chul Choi Eun Kyu Lee 《Biotechnology and Bioprocess Engineering》2005,10(4):309-314
We performed a basic experiment for the rapid, on-line, real-time measurement of hepatitis B surface antigen using a surface
plasmon resonance biosensor. We immobilized anti-HBsAg (hepatitis B surface antigen) polyclonal antibody, as a ligand, to
the dextran layer on a CM5 chip surface that had previously been activated byN-hydroxysuccinimide. A sample solution containing HBsAg was fed through a microfluidic channel, and the reflecting angle change
due to the mass increase from the binding was detected. The binding characteristics between HBsAg and its polyclonal antibody
followed the typical monolayer adsorption isotherm. When the entire immobilized antibody had interacted, no additional, non-specific
binding occurred, suggesting the immunoreaction was very specific. The bound antigen per unit mass of the antibody was independent
of the immobilized ligand density. No significant steric hindrance was observed at an immobilization density of approximately
17.6 ng/mm2. The relationship between the HBsAg concentration in the sample solution and the antigen bound to the ligand was linear up
to ca. 40 μg/mL. This linearity was much higher than that of the ELISA method. It appeared the antigen-antibody binding increased
as the immobilized ligand density increased. In summary, this study showed the potential of this SPR biosensor-based method
as a rapid, simple and multi-sample on-line assay. Once properly validated, it may serve as a more efficient method for HBsAg
quantification for replacing the ELISA. 相似文献
644.
C.T. Höfer S. Di Lella I. Dahmani N. Jungnick N. Bordag S. Bobone Q. Huang S. Keller A. Herrmann S. Chiantia 《生物化学与生物物理学报:生物膜》2019,1861(6):1123-1134
Influenza A virus is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. One of the ten major proteins encoded by the viral genome, the matrix protein M1, is abundantly produced in infected cells and plays a structural role in determining the morphology of the virus. During assembly of new viral particles, M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. The structure of M1 is only partially known. In particular, structural details of M1 interactions with the cellular plasma membrane as well as M1–protein interactions and multimerization have not been clarified, yet.In this work, we employed a set of complementary experimental and theoretical tools to tackle these issues. Using raster image correlation, surface plasmon resonance and circular dichroism spectroscopies, we quantified membrane association and oligomerization of full-length M1 and of different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region, residues 95–105). Furthermore, we report novel information on structural changes in M1 occurring upon binding to membranes. Our experimental results are corroborated by an all-atom model of the full-length M1 protein bound to a negatively charged lipid bilayer. 相似文献
645.
Kaoru Tamada Fumio Nakamura Masateru Ito Xinheng Li Akira Baba 《Plasmonics (Norwell, Mass.)》2007,2(4):185-191
In this short review paper, we summarize some of our ideas to utilize gold nanoparticles for the enhancement of surface plasmon
resonance signals on DNA microarray. The hybridization of target-DNA capped gold nanoparticles with probe DNA on surface provides
ca. ten times stronger optical contrast compared with that of target-DNA molecules. Our simulation result based on the Maxwell-Garnet
theory explains well our experimental data and proves a potential of metallic nanoparticles for the substantial sensitivity
enhancements for biosensor application in DNA diagnostics and bio-affinity studies, which leads to the fabrication of high
resolution DNA microarrays. 相似文献
646.
Label-free technologies, such as surface plasmon resonance, are typically used for characterization of protein interactions and in screening for selection of antibodies or small molecules with preferred binding properties. In characterization, complete binding curves are normally fitted to defined interaction models to provide affinity and rate constants, whereas report points indicative of binding and stability of binding are often used for analysis of screening data. As an alternative to these procedures, here we describe how the analysis, in certain cases, can be simplified by comparison with upper and lower limit binding curves that represent expected or wanted binding profiles. The use of such profiles is applied to the analysis of kinetically complex IgG–Fc receptor interactions and for selection of antibody candidates. The comparison procedure described may be particularly useful in batch-to-batch comparisons and in comparability and biosimilar studies of biotherapeutic medicines. In screening, more informed selections may become possible as entire binding profiles and not a few report points are used in the analysis and as each new sample is directly compared with a predefined outcome. 相似文献
647.
Rosa Cardoso Robert Love Carol L. Nilsson Simon Bergqvist Dawn Nowlin Jiangli Yan Kevin K.‐C. Liu Jing Zhu Ping Chen Ya‐Li Deng H. Jane Dyson Michael J. Greig Alexei Brooun 《Protein science : a publication of the Protein Society》2012,21(12):1885-1896
The heterodimer HIF‐1α (hypoxia inducible factor)/HIF‐β (also known as ARNT‐aryl hydrocarbon nuclear translocator) is a key mediator of cellular response to hypoxia. The interaction between these monomer units can be modified by the action of small molecules in the binding interface between their C‐terminal heterodimerization (PasB) domains. Taking advantage of the presence of several cysteine residues located in the allosteric cavity of HIF‐1α PasB domain, we applied a cysteine‐based reactomics “hotspot identification” strategy to locate regions of HIF‐1α PasB domain critical for its interaction with ARNT. COMPOUND 5 was identified using a mass spectrometry‐based primary screening strategy and was shown to react specifically with Cys255 of the HIF‐1α PasB domain. Biophysical characterization of the interaction between PasB domains of HIF‐1α and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF‐1α and ARNT PasB domains approximately 10‐fold. Detailed NMR structural analysis of HIF‐1α‐PasB‐COMPOUND 5 conjugate showed significant local conformation changes in the HIF‐1α associated with key residues involved in the HIF‐1α/ARNT PasB domain interaction as revealed by the crystal structure of the HIF‐1α/ARNT PasB heterodimer. Our screening strategy could be applied to other targets to identify pockets surrounding reactive cysteines suitable for development of small molecule modulators of protein function. 相似文献
648.
Ganesh Kumar Krishnamoorthy Prashanth Alluvada Shahul Hameed Mohammed Sherieff Timothy Kwa Janarthanan Krishnamoorthy 《Biochemistry and Biophysics Reports》2020
Biophysical techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are routinely used to ascertain the global binding mechanisms of protein-protein or protein-ligand interaction. Recently, Dumas etal, have explicitly modelled the instrument response of the ligand dilution and analysed the ITC thermogram to obtain kinetic rate constants. Adopting a similar approach, we have integrated the dynamic instrument response with the binding mechanism to simulate the ITC profiles of equivalent and independent binding sites, equivalent and sequential binding sites and aggregating systems. The results were benchmarked against the standard commercial software Origin-ITC. Further, the experimental ITC chromatograms of 2′-CMP + RNASE and BH3I-1 + hBCLXL interactions were analysed and shown to be comparable with that of the conventional analysis. Dynamic approach was applied to simulate the SPR profiles of a two-state model, and could reproduce the experimental profile accurately. 相似文献