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SUMMARY:Insight into the Kinetics of Oligomer Formation During Amyloidosis
  - Prof Georges Belfort\, Howard P Isermann Department of Chemical and Bio
 logical Engineering\, Center for Biotechnology and Interdisciplinary Studi
 es\,Rensselaer Polytechnic Institute\, Troy\, USA
DTSTART:20130412T123000Z
DTEND:20130412T133000Z
UID:TALK44222@talks.cam.ac.uk
CONTACT:Dr Myriam Ouberai
DESCRIPTION:Although the critical causative proteins for more than 20 amyl
 oid diseases have been identified\, the process and mechanism by which the
 se proteins induce disease are unknown.  What is known is that these prote
 ins are converted to cross-beta-sheet rich fibril structures called amyloi
 d fibrils.  For over 100 years\, the presence of these fibrils in brain ti
 ssue has been associated with disease (i.e. Alzheimer’s). As a result\, 
 there is a great need to study the thermodynamic\, kinetic and toxic prope
 rties of the oligomer-fiber transition.  We chose human insulin as a model
  amyloid protein for this in vitro study because it is an amyloid protein\
 , it forms fibrils in approx. 3 h at pH 1.6 and 65 C\, exhibits the usual 
 sigmoidal fibril growth curve\, and has been widely used by others.   \nWe
  are mainly interested in both the kinetics and the structural conversion 
 process of native insulin to fibrils and the dissolution of preformed fibr
 ils in order to isolate\, purify and identify the toxic species.  Hence\, 
 we probe the so-called nucleation process for converting a native folded p
 rotein to a beta-sheet rich amyloid fibril and the reverse process.  Using
  a rigorous mechanistic reaction model that incorporates the physical chem
 istry of nucleation and fibril growth dynamics\, we show that the rate con
 stants for nucleation are approx. 10 million times smaller than those for 
 fibril growth (Lee et al. 2007).  During lag phase and prior to the format
 ion of fibrils\, we provide\, using small angle neutron scattering\, consi
 stent evidence of the composition of the insulin nucleus that comprised th
 ree dimers or six monomers (Nayak et al. 2009) and it was likely asymmetri
 c polytetrahedron rather than symmetric octahedron (Meng et al. 2010).  Co
 oling during the lag phase prior to the onset of fibril formation indicate
 d that the oligomers changed linearly with time and that fibril growth was
  slowed at the expense of producing more nuclei (Sorci et al. 2009).  We h
 ave also isolated toxic and non-toxic oligomers from fibril dissolution ex
 periments (Heldt et al. 2011).  Using the final fibril length distribution
 \, we back-calculate for the first time the initial nuclei concentration t
 o be in the range of 20-200 pM (Sorci et al. 2010).  This very low concent
 ration of monomers\, dimers and trimers could explain the difficulty in is
 olating\, detecting and blocking oligomers or nuclei toxicity and the long
  onset time for amyloid diseases (Pease et al. 2010).  The effect of crowd
 ing on kinetics of aggregation will also be discussed (unpublished). \n\nR
 eferences.\nHeldt\, C.L.\, Kurouski\,D.\, Sorci\, M.\, Grafeld\, E.\, Ledn
 ev\, I.K. and Belfort\, G (2011) Isolating toxic insulin amyloid oligomers
  that lack beta-sheets and have wide pH stability\, Biophysical J. 100 (11
 ):2792-800.\nLee\, C. C.\, A. Nayak\, A. Sethuraman\, G. Belfort and G. J.
  McRae (2007). "A three-stage kinetic model of amyloid fibrillation." Biop
 hysical Journal 92(10): 3448-3458.\nMeng\, G.\, N. Arkus\, M. P. Brenner a
 nd V. N. Manoharan (2010). "The free-energy landscape of clusters of attra
 ctive hard spheres." Science 327(5965): 560-3.\nNayak\, A.\, M. Sorci\, S.
  Krueger and G. Belfort (2009). "A universal pathway for amyloid nucleus a
 nd precursor formation for insulin." Proteins-Structure Function and Bioin
 formatics 74(3): 556-565.  \nPease L\, F.\, Sorci\, M.\, Guha\, S.\, Tsai\
 , D. H.\, Zachariah\, M. R.\, Tarlov\, M. J.\, Belfort\, G. (2010) Biophys
 ical J. 99 (12) 3979-85.  \n
LOCATION:MRC Seminar Room M208\, Cavendish Laboratory\, Department of Phys
 ics
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