Thiol modification of diacylglycerol kinase: dependence upon site membrane disposition and reagent hydrophobicity

Czerski, Lech; Sanders, Charles R.

doi:10.1016/s0014-5793(00)01457-5

Cited by 14 publications

(10 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The similarity of the patterns of the rate constants for the reactions of all three reagents with Cys in the tail helix is consistent with all of the substituted Cys reacting only when exposed to water. A similar conclusion was reached about the reactions of disulfide reagents with single-Cys mutants of diacylglycerol kinase in detergent (17). 41 , in which residues -13 to -1 contain a His-tag, and the native sequence starts at M 1 .…”

supporting

confidence: 61%

Reactions of cysteines substituted in the amphipathic N-terminal tail of a bacterial potassium channel with hydrophilic and hydrophobic maleimides

Cortés

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Single cysteine-substitution mutants of KcsA, a K ؉ channel from Streptomyces lividans, were expressed in Escherichia coli, and inner membranes were isolated. The rate constants for the reactions of these cysteines with three maleimides of increasing hydrophobicity, 4-(N-maleimido)phenyltrimethylammonium, N-phenylmaleimide, and N-(1-pyrenyl)maleimide, were determined by back titration of the remaining cysteines with methoxypolyethylene glycol-2-pyridine disulfide (M r 3,000) and quantitation of the fraction of gel-shifted KcsA as a function of reaction time. The patterns of the rate constants for the reactions of all three reagents with eight consecutive cysteines in the partially lipid-immersed amphipathic N-terminal tail helix were the same, with cysteines on the hydrophilic side of the helix reacting faster than Cys on the hydrophobic side. The results are consistent with the tail helix lying with its long axis in the lipid-water interface and with the orientation of the helix fluctuating around this axis. The patterns of the rate constants for the three reagents were similar to the pattern of the probabilities that the substituted cysteines were exposed to water, based on the sum of the free energies of transfer from water to octanol of all of the residues exposed to lipid in each orientation of the helix.I dentification of the surface residues in proteins provides both functional and structural information. The substitutedcysteine accessibility method, which probes engineered cysteines (Cys) with polar reagents directed to water-accessible sulfhydryls (SH), has been widely used to characterize binding sites, channels, and gates (reviewed in ref. 1). It is an indirect approach, in that the reaction of the target Cys is assayed by the resulting irreversible modification of function. Functional assays, especially electrophysiological ones, are highly sensitive and specific and are applicable to a tiny amount of target protein in a sea of other proteins. Most surface residues, however, are not directly involved in function, and reactions of most substituted Cys would not have a detectable functional effect. The general categorization of residues as facing water, facing lipid, or buried in the protein interior requires direct approaches.A direct biophysical approach to surface residues in membrane proteins has been to express single-Cys mutants of the target protein in bacteria, purify the mutants in detergent, attach a SH-directed spin label, reconstitute the labeled protein in membrane, and analyze the mobility and susceptibility of the spin label to hydrophilic and hydrophobic quenchers (reviewed in ref.2). These applications have either preceded a highresolution protein structure or complemented such a structure.Direct biochemical approaches to surface mapping are based on the assay of the reactions of single-Cys substitution mutants with hydrophilic or hydrophobic SH reagents. The reactions have been detected by incorporation of radioactive reagent initially or in a second reaction with remaining SH or, in ...

show abstract

supporting

confidence: 61%

Reactions of cysteines substituted in the amphipathic N-terminal tail of a bacterial potassium channel with hydrophilic and hydrophobic maleimides

Cortés

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Experimental support for the Sukharev & Guy model is provided by the ability to stabilize an open state of MscL through disulfide-bond formation between specifically incorporated cysteines that should be spatially adjacent only in the open state. Due to the role of protein dynamic effects (10) and the influence of the membrane location of the cysteines on the rate of disulfide formation (17), however, it would appear difficult to derive quantitative distance constraints from these experiments. Nevertheless, this general approach has many attractive …”

Section: Mechanosensitive Channels: Msclmentioning

confidence: 99%

The α-Helix and the Organization and Gating of Channels

Spencer

Rees

2002

Annu. Rev. Biophys. Biomol. Struct.

View full text Add to dashboard Cite

The structures of an increasing number of channels and other alpha-helical membrane proteins have been determined recently, including the KcsA potassium channel, the MscL mechanosensitive channel, and the AQP1 and GlpF members of the aquaporin family. In this chapter, the orientation and packing characteristics of bilayer-spanning helices are surveyed in integral membrane proteins. In the case of channels, alpha-helices create the sealed barrier that separates the hydrocarbon region of the bilayer from the permeation pathway for solutes. The helices surrounding the permeation pathway tend to be rather steeply tilted relative to the membrane normal and are consistently arranged in a right-handed bundle. The helical framework further provides a supporting scaffold for nonmembrane-spanning structures associated with channel selectivity. Although structural details remain scarce, the conformational changes associated with gating transitions between closed and open states of channels are reviewed, emphasizing the potential roles of helix-helix interactions in this process.

show abstract

“…Cyanase, a related bacterial enzyme that hydrolyzes cyanate to CO 2 and ammonia, also possesses cysteine moieties that may be easily modified by thiol reagents, leading to the formation of disulfide bonds and an inactive enzyme [78]. Several kinases, such as phosphoglycerate kinase [79], diacylglycerol kinase [80] or creatine-kinase [81] were also shown to be inactivated by thiol reagents such as iodoacetate, Ellman' reagent (5,5'-dithio-bis(2-nitrobenzoic acid)), etc., with inactivation of the enzymes. Such techniques are important for understanding the function of such integral membrane proteins, difficult to be studied by other techniques (such as for instance X-ray crystallography, since it is virtually impossible to crystalize these proteins) [79][80][81].…”

Section: Miscellaneous Applicationsmentioning

confidence: 99%

“…Several kinases, such as phosphoglycerate kinase [79], diacylglycerol kinase [80] or creatine-kinase [81] were also shown to be inactivated by thiol reagents such as iodoacetate, Ellman' reagent (5,5'-dithio-bis(2-nitrobenzoic acid)), etc., with inactivation of the enzymes. Such techniques are important for understanding the function of such integral membrane proteins, difficult to be studied by other techniques (such as for instance X-ray crystallography, since it is virtually impossible to crystalize these proteins) [79][80][81]. The angiotensin II receptor (another trransmembrane protein difficult to be studied by classical techniques) was shown to potentiate ligand binding after treatment with the thiol reagent dithiothreitol [82].…”

Section: Miscellaneous Applicationsmentioning

confidence: 99%

Targeting Cysteine Residues of Biomolecules: New Approaches for the Design of Antiviral and Anticancer Drugs

Scozzafava¹,

Casini

Supuran³

2002

CMC

View full text Add to dashboard Cite

Modification of cysteine (Cys) residues in proteins, due to (i) the participation of the thiol moiety of this amino acid in oxido-reductions reactions; (ii) its ability to strongly coordinate transition metal ions; or (iii) its nucleophilic nature and facile reaction with electrophiles, may be of critical importance for the design of novel types of pharmacological agents. Application of such procedures, recently led to the design of novel antivirals, mainly based on the reaction of zinc finger proteins with disulfides and related derivatives. This approach was particularly successful for developing novel anti-HIV and anti-HPV agents. Several new anticancer therapeutic approaches, mainly targeting tubulin, Ras and fanesyl transferase among others, have also been reported. Miscellaneous other agents/procedures which found less applications for the moment, and which are based on Cys modification reactions, are reviewed. This unique amino acid offers very interesting possibilities to develop particularly useful pharmacological agents, which generally possess a completely different mechanism of action as compared to classical agents in clinical use, avoiding their major problems such as multidrug-resistance of antivirals or antitumor agents or high toxicity associated with classical such chemotherapeutic agents.

show abstract

Thiol modification of diacylglycerol kinase: dependence upon site membrane disposition and reagent hydrophobicity

Cited by 14 publications

References 18 publications

Reactions of cysteines substituted in the amphipathic N-terminal tail of a bacterial potassium channel with hydrophilic and hydrophobic maleimides

Reactions of cysteines substituted in the amphipathic N-terminal tail of a bacterial potassium channel with hydrophilic and hydrophobic maleimides

The α-Helix and the Organization and Gating of Channels

Targeting Cysteine Residues of Biomolecules: New Approaches for the Design of Antiviral and Anticancer Drugs

Contact Info

Product

Resources

About