Low-tech electrophoresis, small but beautiful, and effective: Electrophoretic titration curves of proteins

Gianazza, Elisabetta; Miller, Ingrid; Eberini, Ivano; Castiglioni, Silvia

doi:10.1002/(sici)1522-2683(19990601)20:7<1325::aid-elps1325>3.3.co;2-o

Cited by 3 publications

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“…Transverse urea gradient gel electrophoresis (TUGE), a popular biochemical method, has long been used to investigate the unfolding mechanism of proteins, the existence of folding intermediates, the dissociation and association of proteins (if multimeric in nature), the stability of homologous proteins, etc. , To confirm the unfolding mechanism of His-FKBP22, NTD + , and CTD + , the proteins were analyzed by TUGE following a standard procedure (see Experimental Procedures for details). The gels (Figure ) showed that unfolding of all three proteins produced what are primarily sigmoidal curve-like band patterns with one transition at ∼4–5.5 M urea.…”

Section: Resultsmentioning

confidence: 99%

Domain Structure and Denaturation of a Dimeric Mip-like Peptidyl-Prolyl cis–trans Isomerase from Escherichia coli

et al. 2012

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FKBP22, a protein expressed by Escherichia coli, possesses PPIase (peptidyl-prolyl cis-trans isomerase) activity, binds FK506 (an immunosuppressive drug), and shares homology with Legionella Mip (a virulence factor) and its related proteins. To understand the domain structure and the folding-unfolding mechanism of Mip-like proteins, we investigated a recombinant E. coli FKBP22 (His-FKBP22) as a model protein. Limited proteolysis indicated that His-FKBP22 harbors an N-terminal domain (NTD), a C-terminal domain (CTD), and a long flexible region linking the two domains. His-FKBP22, NTD(+) (NTD with the entire flexible region), and CTD(+) (CTD with a truncated flexible region) were unfolded by a two-state mechanism in the presence of urea. Urea induced the swelling of dimeric His-FKBP22 molecules at the pretransition state but dissociated it at the early transition state. In contrast, guanidine hydrochloride (GdnCl)-induced equilibrium unfolding of His-FKBP22 or NTD(+) and CTD(+) seemed to follow three-step and two-step mechanisms, respectively. Interestingly, the intermediate formed during the unfolding of His-FKBP22 with GdnCl was not a molten globule but was thought to be composed of the partially unfolded dimeric as well as various multimeric His-FKBP22 molecules. Dimeric His-FKBP22 did not dissociate gradually with increasing concentrations of GdnCl. Very low GdnCl concentrations also had little effect on the molecular dimensions of His-FKBP22. Unfolding with either denaturant was found to be reversible, as refolding of the unfolded His-FKBP22 completely, or nearly completely, restored the structure and function of the protein. Additionally, denaturation of His-FKBP22 appeared to begin at the CTD(+).

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Section: Resultsmentioning

confidence: 99%

Domain Structure and Denaturation of a Dimeric Mip-like Peptidyl-Prolyl cis–trans Isomerase from Escherichia coli

et al. 2012

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“…Improved models that properly account for the peptide's three-dimensional structure influence on its net p I values are not yet available. The p I values for peptides can be experimentally determined (as for proteins) by either titration or electrophoretic methods, , but CIEF using standard p I markers is much more convenient since many peptides can be studied in a single separation.…”

Section: Resultsmentioning

confidence: 99%

High-Efficiency Capillary Isoelectric Focusing of Peptides

et al. 2000

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Several approaches are presently being developed for global proteome characterization that are based upon the analysis of polypeptide mixtures resulting from digestion of (often complex) mixtures of proteins. Improved methods for peptide analysis are needed that provide for sample concentration, higher resolution separations, and direct compatibility with mass spectrometry. In this work, methods for the high-efficiency capillary isoelectric focusing (CIEF) separation of peptides have been developed that provide for simultaneous sample concentration and separation according to peptide isoelectric point. Under typical nondenaturing CIEF conditions, peptides are concentrated approximately 500-fold, and peptides present at < 1 ng/ microL were detectable using conventional UV detection. CIEF separations of peptides provided much faster measurements of isoelectric points compared with conventional isoelectric focusing in gels. Very small differences in peptide isoelectric points (deltapI approximately 0.01) could be resolved, High-efficiency CIEF separations for complex peptide mixtures from tryptic digestion of yeast cytosol fractions were obtained and showed significant improvement over those obtained using capillary zone electrophoresis and packed capillary reversed-phase liquid chromatography.

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“…Indeed, IPGs mean today's IEF, and their use is pervasive. CA‐IEF is run less and less often; legendary Ampholine 3.5–10 are not longer on sale, leaving room only for Pharmalyte – a detail for most users, but a detail that is to make almost unfeasible for the few interested to run electrophoretic titration curves 50 on low‐concentration protein solutions due to the heavy background staining of such CAs. Over the years, only three papers have compared results obtained with CA‐IEF and IPGs on individual proteins or whole cellular extracts 51–53.…”

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confidence: 99%

“…Under denaturing conditions (random coil structure), isoelectric point is a sensitive reporter of compositional data 62, 63 and, as outlined in Fig. 3, responds to most point mutations and post‐translational modifications in proteins 50, 64. The p I computed from sequence data is subtly modified in native proteins by the electrostatic influence by nearest neighbors.…”

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Immobilized pH gradients

Gianazza

Righetti

2009

Electrophoresis

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In this short review, we give an account of the steps through which the protocols for operation with IPGs were set up in the early '80s. One of the main achievements by our group was the development of a computer program, pH GRADIENT, for the calculation of pH, buffering power and ionic strength of a mixture of monoprotic buffers titrated within any pH span and for the linearization of the desired pH gradient. Using this program, in 1984 we could devise formulations for IPGs covering up to six pH units, which was the subject of a publication in Electrophoresis (volume 5, pages 88-97). This was the starting point for the use of IPGs for the resolution of protein samples of any composition and for their application as first dimension of 2-DE separations. Currently IPGs are in common use in proteomics investigations, not only along classical protocols but also for sample prefractionation and in shotgun approaches. Much less frequently are they used for 1-DE analytical applications, a field for which in recent years much attention has instead more often focused on capillary electrophoresis/IEF procedures.

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Low-tech electrophoresis, small but beautiful, and effective: Electrophoretic titration curves of proteins

Cited by 3 publications

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Domain Structure and Denaturation of a Dimeric Mip-like Peptidyl-Prolyl cis–trans Isomerase from Escherichia coli

Domain Structure and Denaturation of a Dimeric Mip-like Peptidyl-Prolyl cis–trans Isomerase from Escherichia coli

High-Efficiency Capillary Isoelectric Focusing of Peptides

Immobilized pH gradients

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