Weak-affinity chromatography

Zopf, David; Ohlson, Sten

doi:10.1038/346087a0

Cited by 95 publications

(46 citation statements)

References 4 publications

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“…One way to determine the relative affinity of the N-arm coat protein mutants is to immobilize His-tagged scaffolding protein onto a metal affinity column and use this as a weak-affinity matrix. Through this method, weak affinity between coat and scaffolding proteins can be qualitatively observed (32,37,38). If the N-arm coat protein mutant monomers interact with scaffolding protein, a delay in elution is expected.…”

Section: Resultsmentioning

confidence: 99%

Highly Specific Salt Bridges Govern Bacteriophage P22 Icosahedral Capsid Assembly: Identification of the Site in Coat Protein Responsible for Interaction with Scaffolding Protein

Cortines

Motwani

Vyas

et al. 2014

J Virol

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Icosahedral virus assembly requires a series of concerted and highly specific protein-protein interactions to produce a proper capsid. In bacteriophage P22, only coat protein (gp5) and scaffolding protein (gp8) are needed to assemble a procapsid-like particle, both in vivo and in vitro. In scaffolding protein's coat binding domain, residue R293 is required for procapsid assembly, while residue K296 is important but not essential. Here, we investigate the interaction of scaffolding protein with acidic residues in the N-arm of coat protein, since this interaction has been shown to be electrostatic. Through site-directed mutagenesis of genes 5 and 8, we show that changing coat protein N-arm residue 14 from aspartic acid to alanine causes a lethal phenotype. Coat protein residue D14 is shown by cross-linking to interact with scaffolding protein residue R293 and, thus, is intimately involved in proper procapsid assembly. To a lesser extent, coat protein N-arm residue E18 is also implicated in the interaction with scaffolding protein and is involved in capsid size determination, since a cysteine mutation at this site generated petite capsids. The final acidic residue in the N-arm that was tested, E15, is shown to only weakly interact with scaffolding protein's coat binding domain. This work supports growing evidence that surface charge density may be the driving force of virus capsid protein interactions. IMPORTANCEBacteriophage P22 infects Salmonella enterica serovar Typhimurium and is a model for icosahedral viral capsid assembly. In this system, coat protein interacts with an internal scaffolding protein, triggering the assembly of an intermediate called a procapsid. Previously, we determined that there is a single amino acid in scaffolding protein required for P22 procapsid assembly, although others modulate affinity. Here, we identify partners in coat protein. We show experimentally that relatively weak interactions between coat and scaffolding proteins are capable of driving correctly shaped and sized procapsids and that the lack of these proper protein-protein interfaces leads to aberrant structures. The present work represents an important contribution supporting the hypothesis that virus capsid assembly is governed by seemingly simple interactions. The highly specific nature of the subunit interfaces suggests that these could be good targets for antivirals.

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Section: Resultsmentioning

confidence: 99%

Highly Specific Salt Bridges Govern Bacteriophage P22 Icosahedral Capsid Assembly: Identification of the Site in Coat Protein Responsible for Interaction with Scaffolding Protein

Cortines

Motwani

Vyas

et al. 2014

J Virol

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“…Dissociation constants (K D ) smaller than 10 -5 M make the required on/off-mechanism possible and allow a washing step without significant protein loss [26]. Several TPP binding proteins from microbial organisms, higher plants and animals have already been described [27].…”

Section: Affinity Separation With Crude Cell Brothmentioning

confidence: 99%

Comparison of Purification Efficiencies of Different Affinity Separation Techniques

Koetke¹

2010

toacj

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An affinity separation method for the isolation of recombinantly produced benzoylformiate decarboxylase (BFD) is presented based on the interaction of the enzyme to its cofactor. Enzyme/cofactor interactions are well suited for affinity purification processes with regard to its high affinity and selectivity. Problems can be induced due to harsh desorption conditions that probably diminish the activity of the purified enzyme. The implementation of boronic acids to immobilize the cofactor introduces a gentle release by a simple pH shift from 8.5 to 6. The disadvantages of maminophenyl boronic acids are the high nonspecific adsorption induced by the introduction of a positive charge and a hydrophobic phenyl ring. It was shown that the disadvantages prevail and boronic acid resins are not suitable for protein purification from complex matrices. In contrast to the affinity separation method, where the cofactor was directly immobilized onto the support, a better separation performance could be achieved, which is reflected by a purification factor of 4.

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“…Stable immobilization of suitable model membranes to inert and robust chromatographic media has thus the potential to open up for efficient high-throughput analysis of drug-or biomolecular interactions with membranes. Providing that membrane proteins can be reconstituted into, or adsorbed onto, the immobilized model membrane, systems enabling protein interaction studies by means of techniques such as weak affinity chromatography (WAC) (6) can furthermore be envisioned. WAC, which is emerging as a promising method for the study of weak interactions (K D = mM -M), has previously been successfully used for the study of protein interactions with carbohydrates (7,8), fragment screening (9-11) and for chiral separation (12).…”

Section: Introductionmentioning

confidence: 99%

Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis

et al. 2013

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This is an accepted version of a paper published in Analytical and Bioanalytical Chemistry. This paper has been peer-reviewed but does not include the final publisher proof-corrections or journal pagination.Citation for the published paper: Meiby, E., Morin Zetterberg, M., Ohlson, S., Agmo Hernańdez, V., Edwards, K. (2013 AbstractLipodisks, also referred to as polyethylene glycol (PEG)-stabilized bilayer disks, have previously been demonstrated to hold great potential as model membranes in drug partition studies. In this study, an HPLC-MS system with stably immobilized lipodisks is presented. Functionalized lipodisks were immobilized on two different HPLC support materials either covalently by reductive amination or by streptavidin-biotin binding. An analytical HPLC column with immobilized lipodisks was evaluated by analysis of mixtures containing15 different drug compounds. The efficiency, reproducibility and stability of the system were found to be excellent. In situ incorporation of cyclooxygenase-1 (COX-1) in immobilized lipodisks on a column was also achieved. Specific binding of COX-1 to the immobilized lipodisks was validated by interaction studies with QCM-D. These results taken together open up for the potential to study ligand interactions with membrane proteins by weak affinity chromatography (WAC).

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Weak-affinity chromatography

Cited by 95 publications

References 4 publications

Highly Specific Salt Bridges Govern Bacteriophage P22 Icosahedral Capsid Assembly: Identification of the Site in Coat Protein Responsible for Interaction with Scaffolding Protein

Highly Specific Salt Bridges Govern Bacteriophage P22 Icosahedral Capsid Assembly: Identification of the Site in Coat Protein Responsible for Interaction with Scaffolding Protein

Comparison of Purification Efficiencies of Different Affinity Separation Techniques

Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis

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