222 Pages
English
Gain access to the library to view online
Learn more

Biochemical and NMR characterization of aggregating proteins and their interactions with molecular charperones [Elektronische Ressource] / Saravanakumar Narayanan

-

Gain access to the library to view online
Learn more
222 Pages
English

Informations

Published by
Published 01 January 2004
Reads 25
Language English
Document size 8 MB

Exrait



Department Chemie
der Technischen Universität München



Biochemical and NMR Characterization of Aggregating
Proteins and their Interactions with Molecular Chaperones



Saravanakumar Narayanan



Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität
München zur Erlangung des akademischen Grades eines



Doktors der Naturwissenschaften



genehmigten Dissertation.



Vorsitzende: Univ. Prof. DrSevil Weinkauf

Prüfer der Dissertation: 1. Univ. Prof. Dr. Johannes Buchner
2. Univ. Prof. Dr. Bernd Reif
Humboldt Universität, Berlin.
3. Univ. Prof. Dr. Horst Kessler



Die Dissertation wurde am 17.09.2004 bei der Technischen Universität München
eingereicht und durch die Fakultät für Chemie am 19.10.2004 angenommen.



































f
f
f
f
f
f
f
f
f
f


kbbt[a lay pye[[ekal valbiv[
nbbaq etaza`a '[i[ (tiRkKbq, 1:1:2, ki.M.31)


What Profit have those derived from learning, who worship not the good feet of God
(Thirukkural, 1:1:2, B.C. 31)



















To my family


































f
f
f
I
f
f
f
f
f
<
f
f
f
f
f
f
f
f
f
























klvi kAryil kbpva naqcil
emll niA[kki[ pi]ipl - etqqit[
~rayn tAmvAdy kbpEv neraziyp
paL] KRki[ etainT.
(nalFyaa,2:14:5, ki.pi.300)






Learning has no bounds; the learners days are few. Would they calmly reflect, diseases are many. Let
carefully investigate and make familiar with those things which are essential.
(Naladiyar, 2:14:5, a Tamil ethical literature, A.D 300) Abstract
In this thesis, the mechanism of protein aggregation and the interactions of aggregating
proteins with molecular chaperones are investigated using solution and solid-state NMR spectroscopy.
The influence of chaperones on the protein aggregation is studied for the two systems, Sup35/Hsp104
and A β(1-40)/ αB-crystallin. Protein aggregation itself is investigated using the model systems A β(1-
40) and PI3-SH3.

The yeast prion system (Sup35/Hsp104) was investigated using various NMR (Diffusion,
Saturation Transfer Difference, etc.) and biophysical techniques (Equilibrium dialysis, CD
spectroscopy, etc.). We could show that Hsp104 specifically interacts with critical oligomers of Sup35
and that this interaction modulates the aggregation characteristics.
In the literature it has been shown that the interaction of the Alzheimer’s disease causing
amyloid peptide (A β(1-40)) with a small heat shock protein, αB-crystallin, increases the neurotoxicity
in cultured neurons. We could identify the chemical groups of A β(1-40) that are involved in this
interaction using STD NMR. As a consequence of the interaction, we found that Met35 in A β(1-40)
becomes oxidized. The reason for the increased neurotoxicity is discussed based on these findings.
Using biochemical, NMR and biophysical approaches (Isothermal titration calorimetry, CD
spectroscopy and Colorimetric and a NMR redox assay that developed during this thesis), we could
characterize specific redox properties of A β(1-40) and αB-crystallin. We could show that the
interaction of αB-crystallin with A β(1-40) is copper modulated and redox driven. A simple concept
for the implication with respect to neurotoxicity is presented. For the two chaperone systems, we
observe a general trend for ligand chaperone interactions, which involves redox activity of chaperones.
This is discussed at the end of the thesis.
The molecular mechanism of early steps in fibril formation is studied using the A β(1-40)
peptide. Upon variation of the salt conditions, different oligomeric states of A β(1-40) could be
generated that are analyzed by solution-state NMR experiments. We find that, in addition to specific
peptide-peptide contacts, interactions of anions with selective amino acids of A β(1-40) are key for the
aggregation process.
The SH3 domain of phosphatidylinositol 3'-kinase (PI3-SH3) forms amyloid fibrils when
incubated at low pH for long periods of time. Early stages of fibril formation were studied using
solution-state NMR, whereas the fibril structure was probed by solid-state NMR. By solid-state NMR,
we find that the side chain of H25 is involved in fibril stabilizing interactions. By solution-state NMR ,
we show that modification of PI3-SH3 using Diethyl pyrocarbonate (DEPC) has an influence on the
distribution of soluble and oligomeric PI3-SH3. Even though that the modified protein is more stable,
aggregation is facilitated. Amino acids which specifically affected as a function of pH are identified
1 15using H, N correlation experiments. Structural models for protein aggregation are discussed in the
context of domain swapping of SH3 domains.
IV Parts of this thesis have been or will be published in due course:

1. Saravanakumar Narayanan, Benjamin Bösl, Stefan Walter, Bernd Reif “Importance of
low-molecular weight species for prion propagation in the yeast prion system
Hsp104/Sup35” Proc.Natl.Acad.Sci. USA, (2003) 100 (16), 9286-9291.

2. Salvador Ventura, Jesus Zurdo, Saravanakumar Narayanan, Matildo parreno, Ramon
Mangues, Bernd Reif, F.Xavier Aviles, Christopher Dobson, Luis Serrano “Sequence
triggers for protein aggregation and amyloid formation- An SH3 case”
Proc.Natl.Acad.Sci. USA, (2004) 101 (19), 7258-7263.

3. Saravanakumar Narayanan, Bernd Reif “Characterization of chemical exchange
between soluble and aggregated states of β-Amyloid by solution state NMR upon
variation of salt conditions” Biochemistry, (2004) (In press)


4. Saravanakumar Narayanan, Wilbert Boelens, Wilfried de Jong, Bernd Reif
“Mechanistic understanding of the increased neurotoxicity of β-amyloid induced by the
small heat shock protein αB crystallin” Manuscript prepared for the Submission to
Nature Structural and Molecular Biology.

5. Saravanakumar Narayanan, Stefan Walter, Bernd Reif “Biochemical and structural
investigations of the interactions between the molecular chaperone Hsp104 and the
critical oligomers of yeast prion protein domain of Sup35p” Manuscript prepared for the
Submission to Biochemistry.


6. Saravanakumar Narayanan, Salvador Ventura, Bernd Reif “Solution and solid-state
NMR investigations of mechanism of fibril formation of Pi3-SH3” (in preparation)
V