Folding and assembly of antibodies [Elektronische Ressource] / Matthias Johannes Feige

Technische Universität München
Department Chemie
Lehrstuhl für Biotechnologie



FOLDING AND ASSEMBLY OF ANTIBODIES

Matthias Johannes Feige


Vollständiger Abdruck der von der Fakultät für Chemie der
Technischen Universität München zur Erlangung des akademischen
Grades eines Doktors der Naturwissenschaften genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. M. Groll
Prüfer der Dissertation:
1. Univ.-Prof. Dr. J. Buchner
2. Univ.-Prof. Dr. T. Kiefhaber
3. Univ.-Prof. Dr. S. Weinkauf

Die Dissertation wurde am 28.09.2009 bei der Technischen Universität München
eingereicht und durch die Fakultät für Chemie am 26.11.2009 angenommen.















Etagen, W. Kandinsky, 1929







MIT DEM BEOBACHTEN ERSCHAFFEN WIR.







Table of contents

PART A – INTRODUCTION AND SUMMARY

I. A SHORT INTRODUCTION INTO PROTEIN FOLDING..................................................................... 1
I-1 GENERAL CONSIDERATIONS .............................................................................................................. 1
I-2 A BRIEF HISTORY OF PROTEIN FOLDING.............................................................................................. 1
I-3 THE UNFOLDED STATE ...................................................................................................................... 2
I-3 MOVEMENTS ON THE FREE ENERGY HYPERSURFACE .......................................................................... 4
I-4 THE TRANSITION STATE..................................................................................................................... 4
I-5 PROTEIN FOLDING INTERMEDIATES .................................................................................................... 5
I-6 THE NATIVE STATE............................................................................................................................ 6
I-7 MULTIDOMAIN AND MULTIMERIC PROTEINS ......................................................................................... 7
I-8 THE EVOLUTION OF FOLDABLE POLYPEPTIDES AND FOLDING MECHANISMS........................................... 8
I-9 A SYNOPSIS? ................................................................................................................................... 9
II. ANTIBODY FOLDING AND ASSEMBLY – CLASSICAL THEMES & NOVEL CONCEPTS.......... 11
II-1 PROTEIN FOLDING IN THE ENDOPLASMIC RETICULUM ....................................................................... 11
II-2 A SHORT OVERVIEW OVER ANTIBODY BIOLOGY ................................................................................ 12
II-3 BIOSYNTHESIS OF ANTIBODIES IN THE CELL..................................................................................... 13
II-4 ANTIBODY STRUCTURE AND THE EVOLUTION OF THE IMMUNOGLOBULIN FOLD.................................... 14
II-5 FROM THE FOLDING OF ANTIBODY DOMAINS TO COMPLETE MOLECULES ............................................ 17
II- 6 QUALITY CONTROL OF ANTIBODY FOLDING IN VIVO .......................................................................... 21
II- 7 A COMPREHENSIVE VIEW OF ANTIBODY FOLDING, ASSEMBLY AND QUALITY CONTROL – THE CURRENT
STATUS AND CHALLENGES AHEAD ......................................................................................................... 24
III. A SHORT SUMMARY OF THE WORK........................................................................................... 26
IV. REFERENCES................................................................................................................................. 27



PART B – SCIENTIFIC PUBLICATIONS

V. PUBLISHED SCIENTIFIC PAPER................................................................................................... 38
VI. PUBLISHED BOOK CHAPTERS...................................................................................................106






I. A short introduction into protein folding

I-1 General considerations

Hardly any field of biological research has undergone such considerable changes
and advancements in recent years as has the field of protein folding. Almost five
decades ago the observation that protein folding in the test tube is reversible
(Anfinsen and Haber, 1961) laid the basis of the field. Countless studies on protein
folding have been published since then, and especially in the last few years, the
advent of new techniques has pushed barriers and protein folding emerged as one of
the first truly interdisciplinary fields in biological sciences. This work is supposed to
contribute to this development, in particular by bridging the gap between in silico, in
vitro and in vivo studies on protein folding for a well known model system: antibodies.


I-2 A brief history of protein folding

As soon as it had been appreciated that proteins are irregularly but well defined
structured molecular objects, the question of how proteins could be able to self-
organize began to bother scientists. The adverse effect of this motivation was that the
structural perspective of the protein folding problem shaped its perception. In other
words, early protein folding studies aimed at a well-defined description of the protein
folding phenomenon with only few states involved, unfolded, native and intermediate,
often applying the conceptual framework of organic chemistry developed for small
molecules. The new view of protein folding, despite several shortcomings, has
highlighted this conceptual dilemma of protein folding (Dill and Chan, 1997; Leopold
et al., 1992; Onuchic et al., 1995). Some of its basic ideas had already been
developed earlier and applied to the native state of proteins (Austin et al., 1973;
Frauenfelder et al., 1979; Hartmann et al., 1982). Together they shifted the focus,
and nowadays proteins are regarded as highly dynamical objects. This dynamical
character should not only influence the native state of proteins but in particular be a
dominant factor in folding (Hartmann et al., 1982; Leopold et al., 1992; Henzler-
Wildman et al., 2007a; Henzler-Wildman et al., 2007b). Dynamics implies
1heterogeneity and heterogeneity suggests parallel rather than strictly sequential
pathways for protein folding. This was a main idea of the new view which arose from
a merge of polymer physics and protein science (Dill and Chan, 1997; Leopold et al.,
1992; Onuchic et al., 1995). It put forward that polypeptide chains navigate on a free
energy hypersurface which is generally funnelled towards the native state (Figure I-1).
Accordingly, already few interactions can tip the energetic balance of a polypeptide
chain, predisposing it towards further movement down the funnel and finally the
native state(s) (Dill and Chan, 1997; Onuchic et al., 1995). Even though the funnel
concept alone does not necessarily explain different protein folding phenomena and
is very often applied quite generously, it facilitated novel perspectives on protein
folding as will be outlined in the following.


A B









Figure I-1: A schematic protein folding funnel. Shown are possible folding funnels for a two-state
(A) and a multi-state folder (B). The smoothness and the number and characteristics of the available
energy minima on the free energy hypersurface will shape the folding mechanism. Figure adapted
from (Bartlett and Radford, 2009).


I-3 The unfolded state

Originally, the unfolded state of proteins was termed random coil. This implies the
absence of defined interactions or conformations of an unfolded protein. It is
accordingly expected to behave like a random polymer in a good solvent. This view is
out-dated. It was mainly founded on techniques with too low resolution or too low
sensitivity which could hence only provide global properties for the investigated
2proteins. In contrast, several recent studies on chemically denatured proteins, even
under harsh conditions, have been able to detect significant residual structure, local
and non-local, native and non-native (Crowhurst et al., 2002; Mok et al., 2007;
Sanchez and Kiefhaber, 2003a; Le Duff et al., 2006; Smith et al., 1996). This residual
structure can be expected to have a pronounced effect on the folding free energy
landscape of a protein. If native, it can entropically destabilize the unfolded state
thereby rendering folding more favourable. The classical examples are disulfide
bridges (Pace et al., 1988; Clarke et al., 1995a; Clarke et al., 1995b; Abkevich and
Shakhnovich, 2000; Eyles et al., 1994), which historically also played a significant
role as probes to study protein folding (Creighton, 1974; Creighton, 1975). Non-
covalent interactions, though, seem to be far from absent in unfolded states of
proteins. If non-native, enthalpical stabilization of the unfolded state might be a
consequence. It sho