Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Biophys J
2011 May 18;10010:2513-21. doi: 10.1016/j.bpj.2011.03.063.
Show Gene links
Show Anatomy links
Labeling of specific cysteines in proteins using reversible metal protection.
Puljung MC
,
Zagotta WN
.
???displayArticle.abstract???
Fluorescence spectroscopy is an indispensible tool for studying the structure and conformational dynamics of protein molecules both in isolation and in their cellular context. The ideal probes for monitoring intramolecular protein motions are small, cysteine-reactive fluorophores. However, it can be difficult to obtain specific labeling of a desired cysteine in proteins with multiple cysteines, in a mixture of proteins, or in a protein's native environment, in which many cysteine-containing proteins are present. To obtain specific labeling, we developed a method we call cysteine metal protection and labeling (CyMPL). With this method, a desired cysteine can be reversibly protected by binding group 12 metal ions (e.g., Cd²⁺ and Zn²⁺) while background cysteines are blocked with nonfluorescent covalent modifiers. We increased the metal affinity for specific cysteines by incorporating them into minimal binding sites in existing secondary structural motifs (i.e., α-helix or β-strand). After the metal ions were removed, the deprotected cysteines were then available to specifically react with a fluorophore.
Gaietta,
Multicolor and electron microscopic imaging of connexin trafficking.
2002, Pubmed
Gaietta,
Multicolor and electron microscopic imaging of connexin trafficking.
2002,
Pubmed
Gordon,
Localization of regions affecting an allosteric transition in cyclic nucleotide-activated channels.
1995,
Pubmed
,
Xenbase
Greenfield,
Computed circular dichroism spectra for the evaluation of protein conformation.
1969,
Pubmed
Islas,
Short-range molecular rearrangements in ion channels detected by tryptophan quenching of bimane fluorescence.
2006,
Pubmed
,
Xenbase
Kosower,
Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.
1979,
Pubmed
Kuiper,
A method for site-specific labeling of multiple protein thiols.
2009,
Pubmed
Leverrier,
Metal binding to ligands: cadmium complexes with glutathione revisited.
2007,
Pubmed
Ma,
An extracellular Cu2+ binding site in the voltage sensor of BK and Shaker potassium channels.
2008,
Pubmed
,
Xenbase
Marqusee,
Unusually stable helix formation in short alanine-based peptides.
1989,
Pubmed
Ratner,
A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies.
2002,
Pubmed
Rulísek,
Coordination geometries of selected transition metal ions (Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+) in metalloproteins.
1998,
Pubmed
Sandtner,
In vivo measurement of intramolecular distances using genetically encoded reporters.
2007,
Pubmed
,
Xenbase
Smith,
Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms.
2005,
Pubmed
Spurlino,
The 2.3-A resolution structure of the maltose- or maltodextrin-binding protein, a primary receptor of bacterial active transport and chemotaxis.
1991,
Pubmed
Taraska,
Fluorescence applications in molecular neurobiology.
2010,
Pubmed
Taraska,
Mapping the structure and conformational movements of proteins with transition metal ion FRET.
2009,
Pubmed
Taraska,
Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions.
2009,
Pubmed
Tsien,
The green fluorescent protein.
1998,
Pubmed
Zagotta,
Structural basis for modulation and agonist specificity of HCN pacemaker channels.
2003,
Pubmed
Zheng,
Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry.
2000,
Pubmed