«Mechanisms of De Novo Multi-domain Protein Folding in Bacteria and Eukaryotes Hung-Chun Chang aus Kaohsiung Taiwan, R.O.C. Erklärung Diese ...»
Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München
Mechanisms of De Novo Multi-domain
Protein Folding in Bacteria and Eukaryotes
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 von Herrn Professor Dr. F. Ulrich Hartl betreut.
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfen erarbeitet.
Hung-Chun Chang Dissertation eingereicht am 07. December 2006
1. Gutachter: Professor Dr. F. Ulrich Hartl
2. Gutachter: Professor Dr. Dr. Walter Neupert Mündliche Prüfung am 05. February 2007 Acknowledgements First of all, I would like to express my deepest gratitude to Prof. Dr. F. Ulrich Hartl for giving me the opportunity to study the extremely interesting subject in his laboratory. I would like to thank him for his encouragement and his continual support throughout the entire period of my study. And also many thanks to Dr. Manajit Hayer-Hartl for her numerous helpful discussions and advices.
I would like to thank Prof. Dr. Dr. W. Neupert for his kindly help for correcting my dissertation and being the co-referee of my thesis committee.
Uncountable thanks go to Dr. José M. Barral for his helpful supervision of my work.
His great knowledge and enthusiasm toward science contributed to an invaluable source of inspiration as I worked through the challenges of my project. I would like to thank Dr.
Christian M. Kaiser for fruitful cooperation and many insightful discussions. Their friendships and the good working atmosphere became the main basis for the success of this work.
I thank colleagues in the department of cellular biochemistry for providing accommodative environment to a foreigner like me and many technical helps. In particularly, I would like to thank Dr. Peter Breuer, Dr. Gregor Schaffar and Dr. Andreas Bracher for generously sharing their speciality opinions.
Special thanks to Dr. Ramunas M. Vabulas for his willingness to engage in frequent discussions and his friendship.
I also would like to thank Prof. Dr. F. Ulrich Hartl and Dr. José M. Barral for their intensive review and beneficial feedback on my English writing.
The deepest thanks go to my wife, Yun-Chi Tang, not only for her enormous support and patience, but also beneficial discussions with her. The same deep thanks belong to my parents and my senior brother in Taiwan for their understanding and support.
1. Summary Eukaryotic genomes encode a considerably higher fraction of multi-domain proteins than their prokaryotic counterparts. It has been postulated that efficient co-translational and sequential domain folding has facilitated the explosive evolution of multi-domain proteins in eukaryotes by recombination of pre-existent domains. In the present study, we tested whether eukaryotes and bacteria differ in the folding efficiency and mechanisms of multidomain proteins in general. To this end, a series of recombinant proteins comprised of GFPuv fused to four different robustly folding proteins through six different linkers were generated, and their folding behavior upon expression in E. coli and the yeast S. cerevisiae was compared. Unlike yeast, bacteria were found to be remarkably inefficient at folding these fusion proteins. By following the accumulation of enzymatically active fusion proteins, we found that the rate of appearance of correctly folded fusion protein per ribosome is indeed considerably higher in yeast than in bacteria.
Increasing evidence suggests that elongating polypeptide chains on ribosomes interact substantially with nascent chain binding chaperones to facilitate their folding. Our observations regarding the low efficiency of multi-domain protein folding in bacteria prompted us to investigate the possible roles of trigger factor and DnaK, the two major nascent chain binding chaperones in E. coli, in determining the folding fate of multi-domain nascent polypeptides. For our experiments, we utilized living bacterial strains carrying null deletions of trigger factor and DnaK as well as an in vitro bacterial translation lysate. We found that upon expression under chaperone-depleted conditions, multi-domain proteins such as bacterial β-galactosidase and eukaryotic firefly luciferase fold by a rapid but inefficient default-pathway tightly coupled to translation. Although trigger factor and DnaK improve the folding yield both in vivo and in vitro, these chaperones markedly delay the
factor molecules to translating ribosomes. While β-galactosidase uses this chaperone mechanism effectively, firefly luciferase folding in E. coli remains inefficient. The efficient co-translational domain folding of firefly luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells, thus explaining the higher folding yield of multi-domain proteins in the eukaryotic cytosol.
Additional experiments revealed that efficient protein folding in the eukaryotic model organism S. cerevisiae is supported by a complex chaperone system. Fes1p was structurally and functionally demonstrated to be a nucleotide exchange factor for the yeast cytosolic hsp70 homolog Ssa1p. We first tested the folding of luciferase in a yeast FES1-deletion strain and found that the specific activity of luciferase expressed at elevated temperatures was decreased ~50% compared to the wild-type control. Thus, the folding of luciferase in yeast is dependent on the involvement of Ssa1p in the cytosol. Indeed, without Fes1p, a larger molecular weight species of luciferase could be isolated owing to a longer association of the folding intermediate with Ssa1p and Ydj1p. Additional evidence supporting the notion that the yeast cytosol contains a versatile chaperone network highly efficient in supporting correct protein folding came from the analysis of the fate of recombinantly expressed proteins of bacterial origin in yeast. Our laboratory has previously classified a large number of E. coli proteins based on their chaperone-dependency for folding both in vivo and in vitro. Class I substrates, such as enolase, exhibit low chaperone dependency and accordingly showed only a minor folding deficiency in a yeast YDJ1-deletion strain. Class II substrates, such as DCEA, GATD and SYT, utilize either the DnaK or the GroEL/ES systems for folding and showed a strong inability to fold in the same YDJ1-deletion strain.
Consistent with their stringent requirement for GroEL/ES, class III substrates showed major folding deficiencies even in the wild-type yeast background. Therefore, although the yeast
assistance to proteins with stringent requirements for chaperones not normally found in eukaryotes.
In summary, the present study revealed that the eukaryotic cytosol is capable of folding multi-domain proteins with much higher efficiency than the bacterial cytosol. This can be explained by a presented post-translational folding pathway in bacteria that is enforced by chaperones and is incompatible with co-translational folding of eukaryotic proteins. Thus, a post-translational shift imposed by TF and DnaK on the folding mechanism of multi-domain proteins in bacteria may have profound consequences for the heterologous expression of
2. Introduction Proteins are fundamental to most biological processes. Nearly all the molecular transformations that define cellular metabolism are mediated by protein catalysts. There are structural proteins (molecules of the cytoskeleton, epidermal keratin, viral coat proteins);
catalytic proteins (enzymes); transport and storage proteins (hemoblobin, myoglobin, ferritin); regulatory proteins, (including hormones, many kinases and phosphatases, and proteins that control gene expression); and proteins of the immune system and the immunoglobulin superfamily (including antibodies, and proteins involved in cell-cell recognition and signaling). Proteins also perform regulatory roles, monitoring extracellular and intracellular conditions and relaying information to other cellular components. A complete list of known protein functions would contain many thousands of entries, including proteins that transport other molecules and proteins that generate mechanical and electrochemical forces. And such a list would not account for the thousands of proteins whose functions are not yet fully characterized or, in many cases, are completely unknown.
Clearly, there is considerable validity to the statement that proteins are the “building blocks” of life.
2.1.1. Protein structure In order to cover such a variety of functions, most proteins have to adopt specific and unique three-dimensional structures. Like all polymeric molecules, proteins can be described in terms of levels of organization, in this case, their primary, secondary, tertiary and quaternary structures. A protein’s primary structure is the amino acid sequence of its
structure is the local spatial arrangement of a polypeptide’s backbone atoms without regard to the conformations of its side chains. In most cases, it is referring the formation of helices and sheets from particular regions of a protein. The tertiary structure refers to the threedimensional structure of an entire polypeptide, which is described by the way that helices and sheets are organized and interact in space. As many proteins are composed of two or more polypeptide chains, a protein’s quaternary structure refers to the spatial arrangement of its subunits.
Compared to the thousands of protein tertiary structures that can be found, and the overall uniqueness of conformation of each protein, the protein secondary structures are surprisingly simpler, which include folding patterns such as helices, sheets and turns. The αhelix and the β-sheet are such elements that not only can keep the main chain in an unstrained conformation, but also satisfy the hydrogen-bonding potential of the main-chain N-H and C=O groups. Both patterns were discovered 50 years ago from studies of hair and silk. The first folding pattern to be discovered, the α-helix, was found in the protein αkeratin, which is abundant in skin and its derivatives, such as hair, nails, and horns (Pauling and Corey, 1951a). Within a year of the discovery of the α-helix, a second folded structure, the β-sheet, was found in the protein fibroin, the major constituent of silk (Pauling and Corey, 1951b). These two patterns are particularly common because they result from hydrogen bonding between the N-H and C=O groups in the polypeptide backbone, without involving the side chains of the amino acids. Thus, they can be formed by many different amino acid sequences. In each case, the protein chain adopts a regular, repeating
2.1.2. The protein folding problem The conformation of a single amino acid in a protein can be described by a pair of angles, psi (ψ) and phi (φ) (Figure 1). The peptide bond itself is planar, a consequence of its partial double bond character. ψ describes the angle formed by rotation around the axis through Cα and the carboxyl carbon; φ describes rotation around Cα and the amino group.
Because of steric collisions between atoms within each amino acid, most pairs of ψ and φ angles do not actually occur. G. N. Ramachandran calculated the energy contained in various pairs of ψ and φ angles and found two most stable pairs, the so called α and β conformations (Ramachandran and Sasisekharan, 1968). These two pairs of angles are found to almost exclusively occur naturally in folded proteins, including the two most prominent examples of secondary structure: α-helix and β-strand.
Figure 1. Rotation about bonds in a polypeptide The structure of each amino acid in a polypeptide can be adjusted by rotation about two single bonds.
Phi (Φ) is the angle of rotation about the bond between the nitrogen and the αcarbon atoms, whereas psi (Ψ) is the angle of rotation about the bond between the α-carbon and the carbonyl carbon atoms. The peptide bond is planar as represented in blue shading.
Adapted from (Lehninger et al., 2000).