A new bifunctional spin-label (BSL) has been synthesized that can be immobilized on the surface of proteins, allowing measurement of rotational motion of proteins by saturation-transfer electron paramagnetic resonance (STEPR). The spin-label contains a photoactivatable azido moiety, a cleavable disulfide, and a nitroxide spin with restricted mobility relative to the rest of the label. The label reacts with surface lysine residues modified with beta-mercaptopropionate. Bifunctional attachment is achieved by photoactivation of the azido group. Any spin-label that remains monofunctionally attached after photolysis is removed by reduction of the disulfide. Only bifunctionally attached BSL remains on the protein. Hemoglobin was used to test the utility of the BSL in STEPR by comparison with hemoglobin modified with maleimide spin-label (MSL), a commonly used standard for the STEPR technique. MSL is a monofunctional spin-label which is fortuitously immobilized by local protein structure within hemoglobin. The BSL labeling of hemoglobin did not significantly affect the quaternary structure of hemoglobin as determined by gel filtration chromatography. The conventional EPR spectra of the mono- and bifunctionally attached BSL-hemoglobin were similar to the MSL-hemoglobin spectrum, indicating that both forms of BSL were rigidly bound to hemoglobin. In contrast, the spectrum obtained by reaction of modified hemoglobin lysine residues with MSL indicated that these labels were highly mobile. The monofunctionally attached BSL was mobilized upon octyl glucoside addition whereas bifunctionally attached BSL was only slightly mobilized, suggesting that hydrophobic interactions immobilize the monofunctionally attached label on hemoglobin. The response of STEPR spectra of mono- and bifunctionally attached BSL-hemoglobin to changes in hemoglobin rotational correlation time was similar to the MSL-hemoglobin over the range of 10(-5)-10(-3) s. The spectra of bifunctionally attached BSL indicated slightly less motion than corresponding spectra for MSL or monofunctionally attached BSL. The new BSL is a good reporter of protein rotation and does not require unique protein structures for its immobilization on the protein. Thus, the BSL should be more generally applicable for STEPR studies of membrane protein rotation than existing monofunctional spin-labels.
Download full-text PDF |
Source |
---|---|
http://dx.doi.org/10.1021/bi00476a014 | DOI Listing |
ACS Appl Mater Interfaces
April 2024
Electrochemical Power Sources Division, CSIR-CECRI, Karaikudi 630 003, Tamil Nadu, India.
The reality of long-term rechargeable and high-performance zinc-air batteries relies majorly on cost-effective and eminent bifunctional electrocatalysts, which can perform both the oxygen reduction reaction (ORR) and the oxygen evolution reaction (OER). Herein, we demonstrate a new approach for the synthesis of in-situ-grown layered double hydroxide of iron and cobalt over a cobalt nanoparticle-enriched nitrogen-doped carbon frame (CoL 2:1) by a simple coprecipitation reaction with facile scale-up and explore its electrocatalytic ORR and OER activity for an electrically rechargeable zinc-air battery. Consequently, the developed composite displays excellent ORR and OER activity with an ORR half-wave potential of 0.
View Article and Find Full Text PDFACS Chem Biol
September 2017
Therapeutic Discovery, ‡Neuroscience, and §Pharmacokinetics and Drug Metabolism, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
The voltage-gated sodium channel Na1.7 is a genetically validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain; however functional Na1.
View Article and Find Full Text PDFNat Struct Mol Biol
January 2017
Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
Protein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response that matches ER chaperone capacity to the burden of unfolded proteins. As levels of unfolded proteins decline, a metazoan-specific FIC-domain-containing ER-localized enzyme (FICD) rapidly inactivates the major ER chaperone BiP by AMPylating T518. Here we show that the single catalytic domain of FICD can also release the attached AMP, restoring functionality to BiP.
View Article and Find Full Text PDFACS Appl Mater Interfaces
October 2014
School of Chemical Engineering, Universiti Sains Malaysia, 14300 Nibong Tebal, Penang, Malaysia.
The synthesis of nanocomposite with controlled surface morphology plays a key role for pollutant removal from aqueous environments. The influence of the molecular size of the polyelectrolyte in synthesizing silica-iron oxide core-shell nanocomposite with open shell structure was investigated by using dynamic light scattering, atomic force microscopy, and quartz crystal microbalance with dissipation (QCM-D). Here, poly(diallydimethylammonium chloride) (PDDA) was used to promote the attachment of iron oxide nanoparticles (IONPs) onto the silica surface to assemble a nanocomposite with magnetic and catalytic bifunctionality.
View Article and Find Full Text PDFJ Inorg Biochem
December 2012
Departamento de Química Inorgánica, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain.
The protein shell of apoferritin-encapsulated maghemite nanoparticles was functionalized with two different red-emitting perylenediimide fluorophores (PDI). One glycosacharide-PDI complex has been synthesized for the first time to be labeled to apoferritin-encapsulated maghemite nanoparticles. Bifunctionality of maghemite@perylenediimide was demonstrated by both magnetic-core and fluorescent-labeled shell properties.
View Article and Find Full Text PDFEnter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!