3D struktura membranskih proteinov

TRIDIMENZIONALNA
STRUKTURA MEMBRANSKIH
PROTEINOV
Seminar pri predmetu Biološke membrane
Šolsko leto: 2014/2015
Ljubljana, 15. 1. 2015
Urška Rauter, Špela Tomaž, Valter Bergant in Mirjam Kmetič
Osnovne lastnosti membranskih proteinov
• Strukturo membranskih proteinov
določajo lastnosti membrane.
• α-vijačnice ali β-sodčki.
Slika 1: Bakteriorodpsin iz bakterije Halobacterium salinarum.
PDB koda: 4MD2
Slika 2: OmpA iz bakterije E. coli.
PDB koda: 1BXW
Vpliv membrane na strukturo proteinov
• Hidrofobnost membrane; koncentracija holesterola in sfingomielina.
• Dielektrična konstanta membrane; visoka jakost elektrostatskih interakcij.
• Veliko manj nabitih in polarnih aminokislinskih ostankov.
Slika 3: Transmembranski del proteina GLPH.
a) Prikaz nabitih aminokislinskih ostankov Asp, Glu, Arg in Lys.
b) Prikaz polarnih aminokislinskih ostankov His, Asn in Gln.
• TM heliksi so bogati z glicinom in prolinom.
Slika 4: Transmembranski del proteina GLPH.
f) Z rdečo so označeni C α atomi glicinov in z zeleno so
označeni atomi prolinskega obroča.
Znanstveni razvoj določanja struktur
membranskih proteinov
• 1985: Deisenhofer et. al., struktura fotosintetskega reakcijskega
centra iz bakterije Rhodopseudomonas viridis pri ločljivosti 3 Å.
Slika 4: Stereo risba strukture
fotosintetskega reakcijskega centra.
Slika 5: Število določenih struktur
v odbobju 1985-2008.
Sodobno določanje struktur membranskih proteinov
• Rentgenska kristalografija ostaja najpomembnejša tehnika.
• Priprava kristalov membranskih proteinov je še vedno težavna.
• Poznavanje strukture omogoča razvoj novih zdravil.
• 2012: Nobelova nagrada za kemijo, Lefkowitz in Kobilka, za raziskave na receptorjih sklopljenih z G
proteini.
Slika 6: Ovire v procesu določanja 3D strukture
membranskih proteinov.
Rentgenska kristalografija membranskih
proteinov
ključna pridobitev
kvalitetnih kristalov
membranski proteini
slabo topni v vodi
izolacija s surfaktanti
Kristalizacijske tehnike
• In surfo
PDC – proteindetergent
complex
visok delež surfaktanta
nižja kvaliteta difrakcije
Kristalizacijske tehnike
• In meso
membranski protein
Lipidna
kubična faza
vodni kanali
lipidni dvosloj
majhen delež surfaktanta
kvalitetni, a majhni kristali
Serijska femtosekundna kristalografija
XFEL (X-ray free-electron lasers) – visoka energija
&
femtosekundna kristalografija – obsevanje s kratkimi pulzi
lipidna kubična faza
•
majhni kristali (LCP),
•
zajemanje podatkov pred
uničenjem kristala
•
delo na sobni tempereaturi
Serijska femtosekundna kristalografija
SERIJSKA FEMTOSEKUNDNA
KRISTALOGRAFIJA
VS
SINHROTRON
receptor serotonina 5-HT2B
(z G proteini sklopljen receptor)
Uporaba tehnik NMR pri določevanju struktur
membranskih proteinov
Tekočinski NMR
http://sligarlab.life.uiuc.edu/nanodisc.html
https://commons.wikimedia.org/wiki/File:Liposome_cross_section.png
http://www.cbmn.u-bordeaux.fr/127-research-lipids-in-all-their-states.html
Oriented sample ssNMR
Das et al., 2013
Magic Angle Spinning ssNMR
Park et al., 2012
Beckonert et al., 2010
https://news.slac.stanford.edu/features/phrase-week-magic-angle
Krio-elektronska mikroskopija
Vrsta presevne elektronske mikroskopije
Prednosti
• Slikanje in/ali elektronska
difrakcija
• Nativno stanje proteinov
• Pomoč pri določitvah ali
potrditvah struktur pridobljenih z
drugimi metodami
Omejitve
• Najmanjši delci za opazovanje 300
kDa
• Nizko razmerje signal/ šum
• Poškodbe vzorca
Elektronska kristalografija
• 2D kristalinična razporeditev v dvosloju
• Nezmožnost tvorbe 3D kristalov ali v primeru premajhnih količin
• Nativnejše okolje in lažja tvorba
• 2D kristalov
P. Werten et al. Progress in the analasys of membrane protein structure and function, 2002;
R. Hite et al. Interaction of AQP0 with E. coli lipids,2010
Krio-elektronska tomografija
• Ne moremo očistiti do homogenega vzorca ali pa odstraniti iz
njihovega fiziološkega okolja
• Resolucije do 20 Å
Subramaniam et al. Electron tomography,2003
Single-particle krio-EM
• Mikrograf homogenega vzorca
• Ena najučinkovotejših in enostavnih tehnik
• Pri transmembranskih proteinih zmerne resolucije 10-15 Å
Milne et al. Cryo-electron microscopy,2013; Ludtke et al. Structure of GroEL,2004
Določitev strukture IP3R1 s single-particle
krio-EM
• Inozitol 1,4,5-trifosfatni receptor tipa 1
• 1,3 MDa velik Ca2+ kanalček
• 2002-2004 več struktur pri resolucijah 20-40 Å
Jiang et al. 2002; Serysheva et al. 2003; da Fonesca et al. 2003
Določitev strukture IP3R1 s single-particle
krio-EM
• 2011 pri zmerni resoluciji 10-17 Å
• Začetni vpogled v strukturo in primerjava z drugimi kanalčki
• Zaključitev dolgotrajnih razprav o različnih strukturah
Ludtke et al. Flexible architecture of IP3R1 by cryo-EM, 2011
Viri in literatura
•
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•
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•
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•
4. Rossman, J.S. & Lamb, R.A. (2011). Influenza virus assembly and budding. Virology 411, 229–36
•
5. Belrhali, H., Nollert, P., Royant, A., Menzel, C., Rosenbusch, J.P., Landau, E.M. & Pebay-Peyroula, E. (1999). Protein, lipid and water organization in bacteriorhodopsin crystals: a molecular view of the purple
membrane at 1.9 A resolution. Structure (London, England : 1993) 7, 909–17
•
6. Dong, H., Sharma, M., Zhou, H.-X. & Cross, T.A. (2012). Glycines: role in α-helical membrane protein structures and a potential indicator of native conformation. Biochemistry 51, 4779–89
•
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9. Locher, K.P., Lee, A.T. & Rees, D.C. (2002). The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. Science (New York, N.Y.) 296, 1091–8
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10. Moraes, I., Evans, G., Sanchez-Weatherby, J., Newstead, S. & Stewart, P.D.S. (2014). Membrane protein structure determination - the next generation. Biochimica et biophysica acta 1838, 78–87
•
11. Hopf, T.A., Colwell, L.J., Sheridan, R., Rost, B., Sander, C. & Marks, D.S. (2012). Three-dimensional structures of membrane proteins from genomic sequencing. Cell 149, 1607–21
•
12. Liu, W., Wacker, D., Wang, C., Abola, E. & Cherezov, V. (2014). Femtosecond crystallography of membrane proteins in the lipidic cubic phase. Philosophical transactions of the Royal Society of London. Series B,
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•
13. Blunk, D., Bierganns, P., Bongartz, N., Tessendorf, R. & Stubenrauch, C. (2006). New speciality surfactants with natural structural motifs. New Journal of Chemistry 30, 1705
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14. Caffrey, M., Li, D. & Dukkipati, A. (2012). Membrane protein structure determination using crystallography and lipidic mesophases: recent advances and successes. Biochemistry 51, 6266–88
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15. Landau, E.M. & Rosenbusch, J.P. (1996). Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proceedings of the National Academy of Sciences of the United States of America 93,
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16. Liu, W., Wacker, D., Gati, C., Han, G.W., James, D., Wang, D., Nelson, G., Weierstall, U., Katritch, V., Barty, A., Zatsepin, N.A., Li, D., Messerschmidt, M., Boutet, S., Williams, G.J., Koglin, J.E., Seibert, M.M., Wang, C.,
Shah, S.T.A., Basu, S., Fromme, R., Kupitz, C., Rendek, K.N., Grotjohann, I., Fromme, P., Kirian, R.A., Beyerlein, K.R., White, T.A., Chapman, H.N., Caffrey, M., Spence, J.C.H., Stevens, R.C. & Cherezov, V. (2013). Serial
femtosecond crystallography of G protein-coupled receptors. Science (New York, N.Y.) 342, 1521–4
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