The development of chromatography for the characterization of protein interactions: a personal perspective

2003 ◽  
Vol 31 (5) ◽  
pp. 1010-1014 ◽  
Author(s):  
D.J. Winzor
Keyword(s):  
Ligand Binding ◽  
Protein Interactions ◽  
Equilibrium Constants ◽  
Gel Filtration ◽  
Personal Perspective ◽  
Self Association ◽  
Quantitative Procedure ◽  
Insight Into

This article reviews the progress of a personal endeavour to develop chromatography as a quantitative procedure for the determination of reaction stoichiometries and equilibrium constants governing protein interactions. As well as affording insight into an aspect of chromatography with which many protein chemists are unfamiliar, it shows the way in which minor adaptations of conventional chromatographic practices have rendered the technique one of the most powerful methods available for the characterization of interactions. That pathway towards quantification is followed from the introduction of frontal gel filtration for the study of protein self-association to the characterization of ligand binding by the biosensor variant of quantitative affinity chromatography.

Integrated mass spectrometry-based multi-omics for elucidating mechanisms of bacterial virulence

2021 ◽  
Author(s):  
Lok Man ◽  
William P. Klare ◽  
Ashleigh L. Dale ◽  
Joel A. Cain ◽  
Stuart J. Cordwell
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Large Scale ◽  
Lipid A ◽  
Phenotypic Characterization ◽  
Post Translational Modification ◽  
Form Of Life ◽  
Antimicrobial Interventions

Despite being considered the simplest form of life, bacteria remain enigmatic, particularly in light of pathogenesis and evolving antimicrobial resistance. After three decades of genomics, we remain some way from understanding these organisms, and a substantial proportion of genes remain functionally unknown. Methodological advances, principally mass spectrometry (MS), are paving the way for parallel analysis of the proteome, metabolome and lipidome. Each provides a global, complementary assay, in addition to genomics, and the ability to better comprehend how pathogens respond to changes in their internal (e.g. mutation) and external environments consistent with infection-like conditions. Such responses include accessing necessary nutrients for survival in a hostile environment where co-colonizing bacteria and normal flora are acclimated to the prevailing conditions. Multi-omics can be harnessed across temporal and spatial (sub-cellular) dimensions to understand adaptation at the molecular level. Gene deletion libraries, in conjunction with large-scale approaches and evolving bioinformatics integration, will greatly facilitate next-generation vaccines and antimicrobial interventions by highlighting novel targets and pathogen-specific pathways. MS is also central in phenotypic characterization of surface biomolecules such as lipid A, as well as aiding in the determination of protein interactions and complexes. There is increasing evidence that bacteria are capable of widespread post-translational modification, including phosphorylation, glycosylation and acetylation; with each contributing to virulence. This review focuses on the bacterial genotype to phenotype transition and surveys the recent literature showing how the genome can be validated at the proteome, metabolome and lipidome levels to provide an integrated view of organism response to host conditions.

THE SEPARATION, DETERMINATION, AND CHARACTERIZATION OF 2-AMINO-2-DEOXY-D-GLUCOSE (D-GLUCOSAMINE) AND 2-AMINO-2-DEOXY-D-GALACTOSE (D-GALACTOSAMINE) IN BIOLOGICAL MATERIALS BY GAS–LIQUID PARTITION CHROMATOGRAPHY

1964 ◽  
Vol 42 (4) ◽  
pp. 451-460 ◽  
Author(s):  
M. B. Perry
Keyword(s):  
Hyaluronic Acid ◽  
Chondroitin Sulphate ◽  
Chromatographic Determination ◽  
Partition Chromatography ◽  
Quantitative Analyses ◽  
Optical Rotations ◽  
Trimethylsilyl Derivatives ◽  
Quantitative Procedure

A method for the separation, determination, and characterization of 2-amino-2-deoxy-D-glucose (D-glucosamine) and 2-amino-2-deoxy-D-galactose (D-galactosamine) is presented. Treatment of 2-acetamido-2-deoxy-α-D-glucose and 2-acetamido-2-deoxy-α-D-galactose in pyridine solution with trimethylchlorosilane and hexamethyldisilazane results in a rapid conversion of the glycoses to their respective trimethylsilyl 3,4,6-tri-O-trimethylsilyl-2-acetamido-2-deoxy-α-D-glycosides which are sufficiently stable and volatile to allow their separation and quantitative analysis to be made by gas–liquid partition chromatography. The two trimethylsilyl derivatives, collected by preparative gas–liquid partition chromatography, were crystalline compounds which had sharp melting points and characteristic infrared spectra and specific optical rotations. Quantitative analyses of mixtures of 2-amino-2-deoxy-D-glucose hydrochloride and 2-amino-2-deoxy-D-galactose hydrochloride were made by gas chromatographic analysis of their trimethylsilyl derivatives formed after prior conversion to their N-acetyl derivatives.The analytical procedure was applied to the characterization of 2-amino-2-deoxy-D-glucose in hyaluronic acid and 2-amino-2-deoxy-D-galactose in chondroitin sulphate. The quantitative procedure was also successfully applied to the analysis of mixtures of hyaluronic acid and chrondroitin sulphate by the gas–liquid partition chromatographic determination of the 2-amino-2-deoxy-D-glucose and 2-amino-2-deoxy-D-galactose in the hydrolyzates prepared from synthetic mixtures of the two mucopolysaccharides.

Infrared Characterization and Determination of Self-Association Equilibrium Constants for Methacrylic Acid Copolymers

2000 ◽  
Vol 39 (2) ◽  
pp. 197-223 ◽  
Author(s):  
CHRISTOPHER S. CLEVELAND ◽  
STEPHEN P. FEARNLEY ◽  
YUHONG HU ◽  
MARK E. WAGMAN ◽  
PAUL C. PAINTER ◽  
...  
Keyword(s):  
Methacrylic Acid ◽  
Equilibrium Constants ◽  
Methacrylic Acid Copolymers ◽  
Self Association ◽  
Association Equilibrium

Self-association versus interassociation in hydrogen bonded polymer blends: 1. Determination of equilibrium constants from miscible poly(2,6-dialkyl-4-vinyl phenol) blends

Polymer ◽  
1996 ◽  
Vol 37 (21) ◽  
pp. 4753-4761 ◽  
Author(s):  
Michael M. Coleman ◽  
George J. Pehlert ◽  
Xiaoming Yang ◽  
John B. Stallman ◽  
Paul C. Painter
Keyword(s):  
Polymer Blends ◽  
Equilibrium Constants ◽  
Self Association ◽  
Vinyl Phenol ◽  
Hydrogen Bonded

Self-diffusion of a highly concentrated monoclonal antibody by fluorescence correlation spectroscopy: insight into protein–protein interactions and self-association

Soft Matter ◽  
2019 ◽  
Vol 15 (33) ◽  
pp. 6660-6676 ◽  
Author(s):  
Jessica J. Hung ◽  
Wade F. Zeno ◽  
Amjad A. Chowdhury ◽  
Barton J. Dear ◽  
Kishan Ramachandran ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Protein Interactions ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Protein Protein Interactions ◽  
Fluorescence Correlation ◽  
Self Diffusion ◽  
Self Association ◽  
Insight Into

Measurement and interpretation of self-diffusion of a highly concentrated mAb with different formulations in context of viscosity and protein self-interactions.

The result of equilibrium-constant calculations strongly depends on the evaluation method used and on the type of experimental errors

2001 ◽  
Vol 359 (2) ◽  
pp. 411-418 ◽  
Author(s):  
Hendrik FUCHS ◽  
Reinhard GESSNER
Keyword(s):  
Protein Interactions ◽  
Evaluation Method ◽  
Equilibrium Constants ◽  
Scatchard Analysis ◽  
Protein Protein Interactions ◽  
Data Set ◽  
Slow Kinetics ◽  
Experimental Errors ◽  
Saturation Effects

The determination of equilibrium constants is a widespread tool both to understand and to characterize protein–protein interactions. A variety of different methods, among them Scatchard analysis, is used to calculate these constants. Although more than 1000 articles dealing with equilibrium constants are published every year, the effects of experimental errors on the results are often disregarded when interpreting the data. In the present study we theoretically analysed the effect of various types of experimental errors on equilibrium constants derived by three different methods. A computer simulation clearly showed that certain experimental errors, namely inaccurate background correction, inexact calibration, saturation effects, slow kinetics and simple scattering, can adversely affect the result. The analysis further revealed that, for a given type of error, the same data set can produce different results depending on the method used.

scholarly journals Computer simulation of protein self-association during small-zone gel filtration. Estimation of equilibrium constants

1981 ◽  
Vol 195 (1) ◽  
pp. 213-219 ◽  
Author(s):  
F J Stevens ◽  
M Schiffer
Keyword(s):  
Computer Simulation ◽  
Association Studies ◽  
Equilibrium Constants ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Light Chains ◽  
Immunoglobulin Light Chains ◽  
Bence Jones Protein ◽  
Self Association ◽  
Individual Column

A simulation is developed that qualitatively describes the small-zone-gel-filtration behaviour of a reversibly associating protein. The results reflect the dependence of the apparent molecular weight of a reversibly associating protein on the equilibrium constant (KD) and initial concentration of the protein as well as the column length. The behaviour of a protein on an individual column is characterized and thus a means is provided for estimation of KD. The procedure is extended to describe the behaviour of a mixture of two proteins capable of heterologous as well as homologous association. This computer simulation has been applied in association studies of immunoglobulin light chains [Stevens, Westholm, Solomon & Schiffer (1980) Proc. Natl. Acad. Sci. 77, 1144--1148]. The KD value determined for the Bence--Jones protein Au (10(5) M-1) is close to the value (6.6 X 10(4) M-1) determined by other methods [Maeda, Steffen & Engel (1978) Biophys. Chem. 9, 57-64].

scholarly journals RNA Multimerisation in the DNA Packaging Motor of Bacteriophage φ29

2005 ◽  
Vol 6 (2) ◽  
pp. 127-134 ◽  
Author(s):  
Jonathan P. A. Wood ◽  
Stephanie A. Capaldi ◽  
Mark A. Robinson ◽  
Andrew J. Baron ◽  
Nicola J. Stonehouse
Keyword(s):  
Analytical Ultracentrifugation ◽  
Molecular Level ◽  
Viral Assembly ◽  
Partial Specific Volume ◽  
Wild Type ◽  
Rna Molecules ◽  
Dna Packaging Motor ◽  
Self Association

The use of bacteriophages as experimental tools allows the investigation of interactions between components at the molecular level that are often not possible in more complex virus systems. The bacteriophage φ29 acts as a molecular machine to package its own genomic DNA during viral assembly. Self-associating RNA molecules, called pRNA, have an essential role in the function of this machine. This paper reports the characterization of this self-association (which leads to multimerisation of wild-type and truncated variant pRNAs) by analytical ultracentrifugation (including determination of the partial specific volume of the pRNA), together with an investigation into the domains of the molecule important for multimerisation by the use of complementary DNA probes.

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