Watson, Christopher John (2012) Insulin Analogues for Insulin Receptor Studies and Medical Applications. PhD thesis, University of York.
Abstract
The structure of insulin molecule was determined by Dorothy Hodgkin in 1969.
Subsequently, it has been established that insulin must rearrange upon binding to its
receptor (Insulin Receptor – IR). However, all known structures of the hormone depict its
storage or inactive form. It has been shown that some residues, key for IR binding, are
buried inside the insulin molecule and must be exposed for an efficient insulin-IR complex
formation. It has been postulated that the C-terminal region of the B-chain (~B20-B30) is
dynamic in this process, and that the detachment of the B20-B30 β-strand leads to the
activation of insulin. However, the understanding of the molecular basis of the insulin
regulatory role is hindered by the lack of the structure of the insulin-IR complex; only 3-D
description of the apo-form of the IR ectodomain is known. The very complex molecular
biology behind expression and production of IR fragments also hampers progress in this
field.
In order to facilitate progress towards determination of the insulin-IR complex
crystal structure this work delivered: (i) structural characterisation of highly-active insulin
analogues for stable hormone-IR complexes, (ii) development of various attempts for an
alternative production of L1 domain of human IR, (iii) structural characterisation of the role
of residues B24 and B26 for insulin function, (iv) clarification of individual contributions of
hydrogen bonds stabilising the insulin dimer, (v) understanding of the structural basis of
different functionality of click-chemistry based novel insulin analogues.
This work established that:
(i) the structural signature of the highly active insulin analogues is new -turn at the Cterminus
of the B-chain (the B26 turn) achieved by trans-to-cis isomerisation of the PheB25-
TyrB26 peptide bond. This conformational change exposes residues responsible for IR
binding, (ii) the production of the L1 domain in E. coli, instead of the usual mammalian
expression system, is not feasible, (iii) the structural invariance of the PheB24 is fundamental
to the formation of the insulin-IR complex. It acts as an anchoring and side-chain pivot for
the B26 turn, (iv) removal of the NHB25-COA19 dimer interface hydrogen bond is sufficient
for a complete disruption of the dimer, whilst the other four hydrogen bonds had a less
marked effect in this process, (v) the formation of the B26 turn can be efficiently mimicked
by click-chemistry based intra-B-chain crosslinks.
These results provide a wealth of information about the active form of insulin, they
provide important tools towards the first insulin-IR complex, and deliver novel insulin
analogues.
Metadata
Supervisors: | Brzozowski, A M |
---|---|
Awarding institution: | University of York |
Academic Units: | The University of York > Chemistry (York) |
Identification Number/EthosID: | uk.bl.ethos.570121 |
Depositing User: | Mr Christopher John Watson |
Date Deposited: | 08 Apr 2013 09:54 |
Last Modified: | 08 Sep 2016 13:01 |
Open Archives Initiative ID (OAI ID): | oai:etheses.whiterose.ac.uk:3797 |
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