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Low microwave-ampli...
Low microwave-amplitude ESR spectroscopy : Measuring spin-relaxation interactions of moderately immobilized spin labels in proteins
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- Hedin, Eva M. K. (författare)
- KTH,Bioteknologi
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- Hult, Karl (författare)
- KTH,Biokemi och biokemisk teknologi
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Mouritsen, Ole G. (författare)
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Hoyrup, P. (författare)
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(creator_code:org_t)
- Elsevier BV, 2004
- 2004
- Engelska.
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 60:2, s. 117-138
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- Electron spin resonance (ESR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool for determining protein structure, dynamics and interactions. We report here a method for determining interactions between spin labels and paramagnetic relaxation agents, which is performed under subsaturating conditions. The low microwave-field amplitude employed (h(1) < 0.36 G) only requires standard, commercially available ESR equipment. The effect of relaxation enhancement on the spin-spin-relaxation time, T-2e, is measured by this method, and compared to classical progressive power saturation performed on a free spin label, (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl)methanethiosulfonate (MTSL), and a spin-labeled protein (Thermomyces lanuginosa lipase, TLL-1252C), employing the water-soluble relaxation agent chromium(III) oxalate (Crox) in concentrations between 0-10 mM. The low-amplitude theory showed excellent agreement with that of classical power saturation in quantifying Crox-induced relaxation enhancement. Low-amplitude measurements were then performed using a standard resonator, with Crox, on 11 spin-labeled TLL mutants displaying rotational correlation times in the motional narrowing regime. All spin-labeled proteins exhibited significant changes in T-2e. We postulate that this novel method is especially suitable for studying moderately immobilized spin labels, such as those positioned at exposed sites in a protein. This method should prove useful for research groups with access to any ESR instrumentation.
Ämnesord
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)
Nyckelord
- electron spin resonance
- microwave saturation
- MTSL
- crox
- lipase
- electron-paramagnetic-resonance
- thermomyces-lanuginosa lipase
- bacteriorhodopsin mutants
- progressive saturation
- membrane transporter
- linewidth analysis
- loop region
- dynamics
- liquids
- conformation
- Biochemistry
- Biokemi
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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