Synergy in Protein Engineering

Woods, Ac C.; Guillemette, J. Guy; Parrish, Jc C.; Smith, Michael B.; Wallace, C J A

doi:10.1074/jbc.271.50.32008

Cited by 27 publications

(10 citation statements)

References 37 publications

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“…Cell debris was removed by centrifugation at 13000Ug for 5 min. AMPK was immunoprecipitated from the lysates (100 Wg protein) with an anti-L antibody bound to protein-A Sepharose [17]. Immune complexes were washed with 3U1 ml bu¡er A and AMPK activity determined using the SAMS peptide assay [18].…”

Section: Ampk Activity Measurementsmentioning

confidence: 99%

Protein kinase inhibitors block the stimulation of the AMP‐activated protein kinase by 5‐amino‐4‐imidazolecarboxamide riboside

2002

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The AMP-activated protein kinase (AMPK) is the central component of a protein kinase cascade that plays a major role in energy sensing. AMPK is activated pharmacologically by 5-amino-4-imidazolecarboxamide (AICA) riboside monophosphate (ZMP), which mimics the e¡ects of AMP on the AMPK cascade. Here we show that uptake of AICA riboside into cells, mediated by the adenosine transport system, is blocked by a number of protein kinase inhibitors. Under these conditions, ZMP does not accumulate to su⁄cient levels to stimulate AMPK. Our results demonstrate that careful interpretation is required when using AICA riboside in conjunction with protein kinase inhibitors to investigate the physiological role of AMPK. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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Section: Ampk Activity Measurementsmentioning

confidence: 99%

Protein kinase inhibitors block the stimulation of the AMP‐activated protein kinase by 5‐amino‐4‐imidazolecarboxamide riboside

2002

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“…The development of approaches for the incorporation of unnatural amino acid derivatives into proteins has received considerable attention in recent years 1–9. Several synthetic10 and biosynthetic11 strategies are now available that allow a large number of modifications to be site‐specifically incorporated into proteins.…”

Section: Introductionmentioning

confidence: 99%

Introduction of unnatural amino acids into proteins using expressed protein ligation

et al. 1999

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Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c‐Abl and c‐Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2‐mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine‐containing construct (12) and the ketone‐containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins. © 2000 John Wiley & Sons, Inc. Biopoly 51: 343–354, 1999

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“…Using three positively charged carboxyamidomethyl-methionine sulfonium ion (CAMMS) 1 derivatives 2 ( Fig. 1A) generated from a mutational methionine scan (13), a series of buried mutations at position 52 (Fig. 1B) (14) and the point mutations Y67F, Y67F/ N52I, and Y67F/N52V (15), an attempt to rationalize ⌬E m 0 Ј mut in terms of polarizability of the protein matrix is made.…”

mentioning

confidence: 99%

Protein Matrix and Dielectric Effect in Cytochromec

Blouin

Wallace

2001

Journal of Biological Chemistry

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The effect of the protein matrix on the standard potential of a buried redox center has been investigated by using a selection of mutants and chemical derivatives in Saccharomyces cerevisiae cytochrome c isoform 1. Assuming only local structural perturbation and no alteration of the iron-ligation chemistry, ⌬E m 0 can be regarded as a measure of the difference in polypeptide solvation of the heme charge, which reflects the dielectric properties of the protein. In double mutants Y67F/N52I Y67F/ N52V, where most of the hydrogen bond network in the heme crevice is eliminated, ⌬S redox compares to the wild type. This indicates that a fully consistent hydrogen bond network has a similar polarizability as an apolar matrix. We therefore argue that the variability in net dielectric susceptibility arises from conformational polarizability, a factor that is not a function of atomic properties and coordinates and is therefore hard to predict using conventional physical relationships.There is still much to learn about the factors determining the E m 0 Ј of redox-active proteins. Determinants of E m 0 Ј such as local structural effects (1) as well as the electrostatic landscape of the polypeptide (2) (including the protein's own charges (3, 4), dipolar matrix (5, 6), and surrounding solvent molecules (7)) have already been identified. However, implementation of this knowledge into models does not reliably reproduce experimental observations.The standard redox potential (E m 0 Ј) reflects the thermodynamics of the equilibrium between redox states. The energetics of rearrangement between redox states will therefore reflect the ability of a system to polarize in an electrostatic field. Assuming that the nature of the iron-ligand interaction remains unchanged, the values of ⌬E m 0 Ј of mutation can be regarded as a measure of change in the polarizability, or dielectric response, to the charge of the redox center.With virtually no large redox-dependent conformational change or ligand rearrangement (8 -12), cytochrome c is a suitable model to investigate the contribution of individual residues to the dielectric properties of the protein. Using three positively charged carboxyamidomethyl-methionine sulfonium ion (CAMMS) 1 derivatives 2 (Fig. 1A) generated from a mutational methionine scan (13), a series of buried mutations at position 52 (Fig. 1B) (14) and the point mutations Y67F, Y67F/ N52I, and Y67F/N52V (15), an attempt to rationalize ⌬E m 0 Ј mut in terms of polarizability of the protein matrix is made. EXPERIMENTAL PROCEDURESDetermination of Redox Potential-The measurement of redox potential was made using the method of mixtures (16) in 50 mM potassium phosphate (pH 7.0). The redox state of the sample protein was assayed in a range of redox buffers set by the ratio of ferro/ferricyanide in solution using an E m 0 Ј for the couple of ϩ0.43 V. Thermodynamics of Oxidoreduction-Temperature dependence of the equilibrium between the reduced and oxidized form of the cytochrome c was measured in 50 mM potassium phosphate (pH 7.0) wit...

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Synergy in Protein Engineering

Cited by 27 publications

References 37 publications

Protein kinase inhibitors block the stimulation of the AMP‐activated protein kinase by 5‐amino‐4‐imidazolecarboxamide riboside

Protein kinase inhibitors block the stimulation of the AMP‐activated protein kinase by 5‐amino‐4‐imidazolecarboxamide riboside

Introduction of unnatural amino acids into proteins using expressed protein ligation

Protein Matrix and Dielectric Effect in Cytochromec

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