Editing Site-directed spin labeling (section)

==Spin labeling==
[[Image:Epr spectrum.jpg|thumb|right|400px|EPR spectrum of [[Spin label|proxyl-MTS]] spin labeled yeast [[Cytochrome c|iso-1-cytochrome ''c'']]. [[Spin label]] is attached to [[Cysteine]] 102 residue.]]

Site-directed spin labeling (SDSL) was pioneered in the laboratory of [[Dr. W.L. Hubbell]].<ref>{{cite journal|last=Altenbach|first=C.|author2=Flitsch, S.L. |author3=Khorana, H.G. |author4=Hubbell, W.L. |title=Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines|journal=Biochemistry|year=1989|volume=28|issue=19|pages=7806–7812|doi=10.1021/bi00445a042|pmid=2558712}}</ref><ref>{{cite journal|last=Altenbach|first=C.|author2=Marti, T. |author3=Khorana, H.G. |author4=Hubbell, W.L. |title=Transmembrane Protein Structure: Spin Labeling of Bacteriorhodopsin Mutants|journal=Science|year=1990|volume=248|issue=4959|pages=1088–192|doi=10.1126/science.2160734|pmid=2160734|bibcode = 1990Sci...248.1088A }}</ref>  In SDSL, sites for attachment of spin labels are introduced into recombinantly expressed proteins by [[site-directed mutagenesis]].  [[Functional group]]s contained within the spin label determine their specificity.  At neutral pH, protein thiol groups specifically react with the functional groups methanethiosulfonate, maleimide, and iodoacetamide, creating a covalent bond with the amino acid [[cysteine|Cys]].<ref name="Klare2009">{{cite journal|last=Klare|first=J.P.|author2=Steinhoff, H.-J. |title=Spin Labeling EPR|journal=Photosynthesis Research|year=2009|volume=102|issue=2–3|pages=377–390|doi=10.1007/s11120-009-9490-7|pmid=19728138|s2cid=40673871}}</ref>   
Spin labels are a unique molecular reporter, in that they are paramagnetic (contain an unpaired electron).  Spin labels were first synthesized in the laboratory of [[H. M. McConnell]] in 1965.<ref>{{cite journal|last=Stone|first=T.J.|author2=Buckman, T. |author3=Nordio, P.L. |author4=McConnell, H.M. |title=Spin-labeled biomolecules|journal=Proc. Natl. Acad. Sci. USA|year=1965|volume=54|issue=4|pages=1010–1017|pmid=5219813|pmc=219782|doi=10.1073/pnas.54.4.1010 |bibcode=1965PNAS...54.1010S|doi-access=free}}</ref>  Since then, a variety of nitroxide [[spin label]]s have enjoyed widespread use for the study of macromolecular structure and dynamics because of their stability and simple [[Electron spin resonance|EPR]] signal. 
The nitroxyl radical (N-O) is usually incorporated into a heterocyclic ring (e.g. [[pyrrolidine]]), and the unpaired electron is predominantly localized to the N-O bond.  Once incorporated into the protein, a spin label's motions are dictated by its local environment.  Because spin labels are exquisitely sensitive to motion, this has profound effects on its EPR spectrum.<ref name="Klare2009" /><ref>{{cite journal|last=Bordignon|first=E.|author-link=Enrica Bordignon|author2=Steinhoff, H.-J. |title=Membrane protein structure and dynamics studied by site-directed spin labeling ESR|journal= In Biological Magnetic Resonance 27 - ESR Spectroscopy in Membrane Biophysics|year=2007|volume=27|series=Biological Magnetic Resonance|pages=129–164|doi=10.1007/978-0-387-49367-1_5|isbn=978-0-387-25066-3}}</ref>

The assembly of multi-subunit membrane protein complexes has also been studied using spin labeling. The binding of the PsaC subunit to the PsaA and PsaB subunits of the photosynthetic reaction center, Photosystem I, has been analyzed in great detail using this technique.<ref>{{cite journal|last1=Jagannathan|first1=B|last2=Dekat|first2=S|last3=Golbeck|first3=JH|last4=Lakshmi|first4=KV|title=The Assembly of a Multisubunit Photosynthetic Membrane Protein Complex: A Site-Specific Spin Labeling EPR Spectroscopic Study of the PsaC Subunit in Photosystem I|journal=Biochemistry|date=2010|volume=49|issue=11|pages=2398–2408|doi=10.1021/bi901483f|pmid=20158221}}</ref>

Dr. Ralf Langen's group showed that SDSL with EPR (University of Southern California, Los Angeles) can be used to understand the structure of amyloid fibrils and the structure of membrane bound Parkinson's disease protein alpha-synuclein.<ref>{{cite journal|last=Chen|first=M.|author2=Margittai, M. |author3=Chen, J. |author4=Langen, R. |title=Investigation of alpha-Synuclein Fibril Structure by Site-directed Spin Labeling|journal=J. Biol. Chem.|year=2007|volume=282|issue=34|pages=24970–24979|doi=10.1074/jbc.M700368200 |pmid=17573347|doi-access=free}}</ref> A 2012 study generated a high resolution structure of IAPP fibrils using a combination of SDSL, pulse EPR and computational biology.<ref>{{cite journal|last=Bedrood|first=Sahar|author2=Yiyu Li |author3=J. Mario Isas |author4=Balachandra G. Hegde |author5=Ulrich Baxa |author6=Ian S. Haworth |author7=Ralf Langen |title=Fibril Structure of Human Islet Amyloid Polypeptide|pmid=22187437|doi=10.1074/jbc.M111.327817|volume=287|issue=8|date=February 2012|journal=J. Biol. Chem.|pages=5235–41|pmc=3285303|doi-access=free}}</ref> 
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