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Folding and Assembly of Antibodies 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 1 heterogeneity 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 2 proteins. 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 should be kept in mind, however, that most studies on the unfolded state of proteins dealt with rather harsh conditions of elevated temperature or high denaturant concentrations. Under physiological conditions, the amount of structure in unfolded states of proteins can be expected to be significantly higher as have revealed the few accessible cases (Religa et al., 2005; Marsh et al., 2007; Chugha et al., 2006) and a compaction of chemically unfolded proteins with lowering denaturant concentrations suggest (Sherman and Haran, 2006; Merchant et al., 2007). Even more important, in vivo, proteins encounter quite a different scenario than dilution from a chemically denatured state. They begin their folding process often during translation or translocation; processes, which only make parts of a protein at a certain time amenable for folding, and in particular to form non-local interactions
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