Characterization Of Protein Binding Research Articles

Characterization of Protein Binding on an Extracorporeal Membrane Oxygenation (ECMO) Circuit Following the Priming Procedure.

Characterization of Protein Binding on an Extracorporeal Membrane Oxygenation (ECMO) Circuit Following the Priming Procedure.

Studying protein-protein interactions: progress, pitfalls and solutions.

Signalling proteins are intrinsic to all biological processes and interact with each other in tightly regulated and orchestrated signalling complexes and pathways. Characterization of protein binding can help to elucidate protein function within signalling pathways. This information is vital for researchers to gain a more comprehensive knowledge of cellular networks which can then be used to develop new therapeutic strategies for disease. However, studying protein-protein interactions (PPIs) can be challenging as the interactions can be extremely transient downstream of specific environmental cues. There are many powerful techniques currently available to identify and confirm PPIs. Choosing the most appropriate range of techniques merits serious consideration. The aim of this review is to provide a starting point for researchers embarking on a PPI study. We provide an overview and point of reference for some of the many methods available to identify interactions from in silico analysis and large scale screening tools through to the methods used to validate potential PPIs. We discuss the advantages and disadvantages of each method and we also provide a workflow chart to highlight the main experimental questions to consider when planning cell lysis to maximize experimental success.

Multimodal chromatography: Characterization of protein binding and selectivity enhancement through mobile phase modulators

The unique selectivity of mixed mode chromatography resins is driving increasing utilization of these novel selectivities into bioprocess applications. There is a need for improved fundamental understanding of protein binding to these stationary phases to enable the development of efficient and robust purification processes. A panel of four monoclonal antibodies and two model proteins were employed to characterize protein interaction with a mixed-mode chromatographic resin comprising a hydrophobic ligand with cation-exchange functionality. Binding of these proteins was studied as a function of salt concentration and pH in the presence of various mobile phase modulators. This knowledge was applied towards screening mobile phase modulators that could selectively decrease host cell protein levels during monoclonal antibody purification.

Saturation-Transfer Difference (STD) NMR: A Simple and Fast Method for Ligand Screening and Characterization of Protein Binding

Saturation transfer difference (STD) NMR has emerged as one of the most popular ligand-based NMR techniques for the study of protein−ligand interactions. The success of this technique is a consequence of its robustness and the fact that it is focused on the signals of the ligand, without any need of processing NMR information about the receptor and only using small quantities of nonlabeled macromolecule. Moreover, the attractiveness of this experiment is also extendable to the classroom. In the context of a practical NMR class, this experiment is ideal to illustrate some fundamental NMR concepts, such as the nuclear Overhauser effect and relaxation in a multidisciplinary context, bridging chemistry and biochemistry with a taste of medicinal chemistry.We use the readily available human serum albumin (HSA), 6-d,l-methyl-tryptophan (6-CH3-Trp), and 7- d,l-methyl-tryptophan (7-CH3-Trp) to introduce the STD-NMR experiment and to illustrate its applicability for ligand screening, mapping of binding moieties, and determination of the dissociation constant, in a context that can be explored or adapted to the student’s course level and topic (chemistry or biochemistry). We also cover the most important theoretical aspects of the STD experiment, calling attention to some of its limitations and drawbacks without a complex theoretical approach.

Mass spectrometric analysis of ubiquitin–platinum interactions of leading anticancer drugs: MALDI versus ESI

The protein binding of anticancer metallodrugs is regarded as an important part in their mode of action both for delivering the active moiety into the tumor but also being responsible for deactivation and/or unwanted side effects. Characterization of protein binding and release may allow new drugs to be designed which are devoid of protein interactions or capable of binding selectively to protein targets. Herein, we report the comparison of different ionization techniques, i.e. matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray ionization mass spectrometry (nESI-MS), for the analysis of small protein–platinum anticancer drug interactions. For this purpose, cisplatin, transplatin and oxaliplatin were incubated with the model proteinubiquitin (Ub) at a molar ratio of 2 : 1 (Pt : Ub) followed by MS analysis. Cisplatin, transplatin and oxaliplatin formed mainly monoadducts with Ub, but of significantly different composition. As reported earlier, cisplatin forms mainly bifunctional Ub–[Pt(NH3)2] adducts, while with transplatin the most abundant adduct was found to be a monofunctional Ub–[Pt(NH3)2Cl] species. Oxaliplatin formed exclusively bifunctional species of the formula Ub–[Pt(chxn)] (chxn = cyclohexane-1,2-diamine). The applied analysis methods provide comparable results. However, the higher resolution of the nESI-quadrupole time-of-flight (QToF)-MS allowed unambiguous characterization of a series of mono- and bis-adducts including Ub–[Pt(NH3)2(H2O)] for both cisplatin and transplatin. Applying nESI-ion trap (IT)-MS showed the advantage of higher sensitivity than the ToF instruments, allowing the detection of bisadducts of oxaliplatin after one week of incubation. In contrast to the ESI mass spectra, MALDI showed a higher degree of fragmentation of the Ub–platinum conjugates.