Mathematical models are now gaining in importance for descriptions of biological processes. In this thesis, such models have been used to identify and analyze principles that govern bacterial protein synthesis under amino acid limitation. New techniques, that are generally applicable for analysis of intrinsic fluctuations in systems of chemical reactions, are also presented. It is shown how multi-substrate reactions, such as protein synthesis, may display zero order kinetics below saturation, because an increase in one substrate pool is compensated by a decrease in another, so that the overall flow is unchanged. Under those conditions, metabolite pools display hyper sensitivity and large fluctuations, unless metabolite synthesis is carefully regulated. It is demonstrated that flow coupling in protein synthesis has consequences for transcriptional control of amino acid biosynthetic operons, accuracy of mRNA translation and the stringent response. Flow coupling also determines the choices of synonymous codons in a number of cases. The reason is that tRNA isoacceptors, cognate to the same amino acid, often read different codons and become deacylated to very different degrees when their amino acid is limiting for protein synthesis. This was demonstrated theoretically and used to successfully predict the choices of control codons in ribosome mediated transcriptional attenuation and codon bias in stress response genes. New tools for the analysis of internal fluctuations have been forged, most importantly, an efficient Monte Carlo algorithm for simulation of the Markov-process corresponding to the reaction-diffusion master equation. The algorithm makes it feasible to analyze stochastic kinetics in spatially extended systems. It was used to demonstrate that bi-stable chemical systems can display spontaneous domain separation also in three spatial dimensions. This analysis reveals geometrical constraints on biochemical memory circuits built from bistable systems. Further, biochemical applications of the Fokker-Planck equation and the Linear Noise Approximation have been explored.

Although platelets are anucleate, they do inherit 1500-3000 mRNA transcripts from their megakaryocyte progenitors, in addition to all the machinery essential for protein synthesis; however, there is little understanding why platelets initiate de novo synthesis of these transcripts. Our group demonstrated that fibrinogen (Fg), a ligand of platelet Glycoprotein (GP)IIb-IIIa (αIIbβ3 integrin), is required for platelet P-selectin expression and that engagement of Fg with GPIIb-IIIa is essential for this process. The present study shows that murine platelets incubated with Fg synthesize P-selectin de novo, and this synthesis is blocked by puromycin. A similar effect is also observed when platelets are incubated with fibronectin, another ligand of GPIIb-IIIa. Furthermore, platelets from both ligand- (Fg−/−, von Willebrand factor−/−, apolipoprotein A-IV−/−) and GPIIb-IIIa-deficient mice have altered proteomes. These data suggest an intricate mechanism by which engagement of platelets with their environment triggers signal-dependent translation of the platelet transcriptome, consequently altering the platelet proteome.

Although platelets are anucleate, they do inherit 1500-3000 mRNA transcripts from their megakaryocyte progenitors, in addition to all the machinery essential for protein synthesis; however, there is little understanding why platelets initiate de novo synthesis of these transcripts. Our group demonstrated that fibrinogen (Fg), a ligand of platelet Glycoprotein (GP)IIb-IIIa (αIIbβ3 integrin), is required for platelet P-selectin expression and that engagement of Fg with GPIIb-IIIa is essential for this process. The present study shows that murine platelets incubated with Fg synthesize P-selectin de novo, and this synthesis is blocked by puromycin. A similar effect is also observed when platelets are incubated with fibronectin, another ligand of GPIIb-IIIa. Furthermore, platelets from both ligand- (Fg−/−, von Willebrand factor−/−, apolipoprotein A-IV−/−) and GPIIb-IIIa-deficient mice have altered proteomes. These data suggest an intricate mechanism by which engagement of platelets with their environment triggers signal-dependent translation of the platelet transcriptome, consequently altering the platelet proteome.

B virus (Cercopithecine herpesvirus 1), which causes an often fatal zoonotic infection in humans, shares extensive homology with human herpes simplex virus type 1 (HSV-1). The ƒ×134.5 gene of HSV-1 plays a major role in counteracting dsRNA-dependent protein kinase (PKR) activity. HSV-1 Us11 protein, if expressed early as a result of mutation, binds to PKR and prevents PKR activation. The results of experiments in this dissertation revealed that although B virus lacks a ƒ×134.5 gene homolog, it is able to inhibit PKR activation, and subsequently, eIF2ƒÑ phosphorylation. The initial hypothesis was that B virus Us11 protein substitutes for the function of ƒ×134.5 gene homolog by blocking cellular PKR activation. Using western blot analysis, Us11 protein (20 kDa) of B virus was observed early following infection (3 h post infection). Expression of B virus Us11 protein was not blocked by phosphonoacetic acid (PAA), an inhibitor of DNA replication, confirming Us11 is not a ¡§true late¡¨ gene of B virus as it is in HSV-1. Analysis of these results suggested that B virus Us11 protein compensates for the lack of the ƒ×134.5 gene homolog and prevents PKR activation. Next, the results demonstrated that B virus Us11 recombinant protein prevented PKR activation by dsRNA in vitro. A B virus Us11 protein stable expression cell line (U373-BVUs11) was established to investigate whether Us11 protein inhibited PKR activation in vivo. Experiments revealed that B virus Us11 protein stably expressed in U373 cells prevented PKR activation and subsequent eIF2ƒÑ phosphorylation induced by the infection of these cells with ƒ´ƒ×134.5 of HSV-1. As the consequence of preventing PKR activation and subsequent eIF2ƒÑ phosphorylation, B virus Us11 protein complemented ƒ´ƒ×134.5 HSV-1 in U373 cells as evidenced by restoration of virus protein synthesis and replication in U373 cells. Furthermore, pull-down assays showed that B virus Us11 protein binds to PKR. In addition, the results demonstrated that B virus Us11 protein stably expressed in U373 cells counteracted the inhibiting effect of IFN-ƒÑ on HSV-1 replication by preventing PKR activation. These data suggested that B virus and HSV-1, two closely related viruses, use different mechanisms to counteract PKR activity.

The purpose of this thesis is to analyze the variation in the maximal discrimination of the interaction between cognate and a non-cognate codon and anti-codon (also called the d-value). The variation was analyzed with a multiple regression model with the d-value as the dependent variable and with the codon position and the different mRNA and tRNA bases as independent variables. The result of the analysis not only confirmed earlier studies that the maximal accuracy was highest in the second codon position and lowest in the third codon position but we also found significant relationships and interaction effects.

The field of protein engineering has been greatly augmented by the expansion of the genetic code using unnatural amino acids as well as the development of cell-free synthesis systems with high protein yield. Cell-free synthesis systems have improved considerably since they were first described almost 40 years ago. Residue specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an amino acid depleted cell-free protein synthesis system that can be used to study residue specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines high protein expression yields with a high level of analog substitution in the target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid-depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo format. We use this amino acid depleted cell-free synthesis system for the directed evolution of streptavidin, a protein that finds wide application in molecular biology and biotechnology. We evolve streptavidin using in vitro compartmentalization in emulsions to bind to desthiobiotin and find, at the conclusion of our experiment, that our evolved streptavidin variants are capable of binding to both biotin and desthiobiotin equally well. We also discover a set of mutations for streptavidin that are potentially powerful stabilizing mutations that we believe will be of great use to the greater research community. / text

Η κλινδαμυκίνη αποτελεί μέλος της οικογενείας των MLS αντιβιοτικών, με ευρύτατες εφαρμογές στην Ιατρική. Προσδεδενόμενη στο κέντρο της πεπτιδυλοτρανσφεράσης, δρα ως αναστολέας της πρωτεϊνικής σύνθεσης. Η ακριβής θέση δράσης της δεν έχει πλήρως διασαφηνισθεί. Διάφορες μελέτες έχουν δείξει πως δρα στην Α θέση, ενώ άλλες στην Ρ θέση της μεγάλης ριβοσωματικής υπομονάδας. Στην παρούσα εργασία γίνεται λεπτομερής κινητική ανάλυση της αναστολής του σχηματισμού του πεπτιδικού δεσμού από την κλινδαμυκίνη σε ιοντικό περιβάλλον που πλησιάζει το φυσιολογικό του κυττάρου (4,5 mM Mg2+, 150 mM NH4+). Συγκεκριμένα, η δράση της κλινδαμυκίνης μελετήθηκε σε ένα σύστημα ελεύθερο-κυττάρων του εντεροβακτηρίου Escherichia coli, όπου ένας πεπτιδικός δεσμός σχηματίζεται μεταξύ πουρομυκίνης και AcPhe-tRNA, προσδεδενόμενου στην Ρ-θέση ριβοσωμάτων προγραμματισμένων με poly(U). Η πουρομυκίνη δρα ως ανάλογο του 3΄ άκρου ενός αμινοακυλο-tRNA ενώ το τριμερές AcPhe-tRNA∙ poly(U)∙ ριβόσωμα, σύμπλοκο C, ως ανάλογο του εναρκτήριου μεταφραστικού συμπλόκου. Η κινητική ανάλυση αποκάλυψε ότι η κλινδαμυκίνη συμπεριφέρεται ως αναστολέας βραδείας δεσμεύσεως. Μετά από μια παροδική αλληλεπίδραση με την Α-θέση, εγκαθίσταται κοντά στην Ρ-θέση του ριβοσώματος με αποτέλεσμα να επηρεάζει την ταχύτητα σχηματισμού του πεπτιδικού δεσμού. Στην συνέχεια μελετήθηκε η επίδραση των πολυαμινών στην αλληλεπίδραση της κλινδαμυκίνης με το σύμπλοκο C. Τα αποτελέσματα έδειξαν πως η σπερμίνη παρεμποδίζει την επίδραση της κλινδαμυκίνης στην Ρ-θέση, όμως επηρεάζει ευνοϊκά και σε μεγαλύτερο βαθμό, την αρχική δέσμευση του φαρμάκου στην Α-θέση ελαττώνοντας το εντροπικό κόστος. Η επίδραση αυτή δεν μεταβλήθηκε όταν αντί της σπερμίνης χρησιμοποιήθηκαν ριβοσώματα επισημασμένα μ’ένα φωτοδραστικό ανάλογο της σπερμίνης, την Ν1-αζιδοβενζαμιδινο-σπερμίνη, ή όταν στο διάλυμα επώασης προστέθηκε μείγμα σπερμίνης και σπερμιδίνης. Πειράματα σταυρο-σύνδεσης έδειξαν ότι η σπερμίνη προσδένεται πλησίον της θέσης δέσμευσης της κλινδαμυκίνης. Η παρατήρηση αυτή οδήγησε στην υπόθεση, ότι οι πολυαμίνες δεσμευόμενες πλησίον της θέσης πρόσδεσης της κλινδαμυκίνης επηρεάζουν την αλληλεπίδραση του αντιβιοτικού με το ριβόσωμα, επάγοντας αλλαγές διαμόρφωσης στο ριβοσωμικό σύμπλοκο. Η υπόθεση αυτή βρίσκεται σε συμφωνία με πειράματα χημικής προστασίας, που έδειξαν, ότι οι πολυαμίνες επηρεάζουν σημαντικά την τριτοταγή δομή του ριβοσώματος. Από άποψη φαρμακευτικών εφαρμογών η παρούσα μελέτη εισηγείται ότι, κάθε φορά που ένα αντιβιοτικό, με μοριακό στόχο το ριβόσωμα, είναι προς σχεδιασμό, η επίδραση του ιοντικού περιβάλλοντος πρέπει να λαμβάνεται σοβαρά υπόψη. / Clindamycin is a representative antibiotic of the MLS family, widely used in clinical practice. It inhibits protein synthesis by binding to the peptidyltransferase center of the ribosome. Clindamycin’s exact site of action is not known. Several studies have shown that it is an inhibitor of the A-site. However, there are also stydies that suggest that clindamycin acts at the P-site of the large ribosomal subunit. In this study, we re-examined the mechanism by which the antibiotic inhibits the formation of peptide bond in an ionic environment that resembles in vivo conditions (4.5 mM Mg2+, 150 mM NH4+). Clindamycin was investigated in a cell-free system derived from Escherichia coli, in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly (U) –programmed ribosomes. Puromycin can be considered as an analogue of the 3’-end of aminoacyl-tRNA, while the ternary complex AcPhe-tRNA∙poly(U)∙ ribosome (complex C) as an analogue of the initiation translation complex. Kinetics revealed that clindamycin behaves as a slow – binding inhibitor. After a transient interaction with the A-site of ribosomes, it slowly accommodates near the P-site so that peptide bond is still formed but with a lower velocity. Next, we investigated the influence of polyamines to the interaction of clindamycin with complex C. It was found that spermine hinders the accommodation of clindamycin to the P-site, but exerts a beneficial, more pronounced, effect on the potency of the drug by lowering the entropic cost of clindamycin binding to the A-site. Polyamine effect was not substantially altered when ribosomes labeled with a photoreactive analogue of spermine, N1-azidobenzamidino-spermine, were used or when a mixture of spermine and spermidine was added in the incubation mixture, instead of spermine alone. Cross-linking experiments have demonstrated that spermine binds to the vicinity of the antibiotic binding pocket. This observation temped us to suppose that polyamines bound adjacently to the binding site of clindamycin modulate the interaction of this drug with the ribosome by inducing conformational changes in the elongating ribosomal complex. Such a hypothesis is in agreement with chemical protection data revealing that polyamines influence significantly the tertiary structure of ribosomes. From the stand point of pharmaceutical applications, the present work postulates that when a drug is designed to target to the ribosome, the influence of the ionic environment should be taken into account.

Many viruses can produce multiple proteins from a single mRNA sequence by encoding the proteins in overlapping genes. One mechanism that causes the ribosomes of infected cells to decode both genes is –1 programmed ribosomal frameshifting. In this process, structural elements of the viral mRNA signal the ribosome to shift reading frames at a specific point. Although –1 frameshifting has been recognized since 1985, the mechanism is not well understood. I have developed a stochastic model of mRNA translation that includes the possibility of a –1 frameshift at any codon. The transition probabilities between states of the model are based on the energetics of local molecular interactions. The model reproduces observed translation rates as well as both the location and efficiency of frameshift events in the HIV-1 gag-pol sequence. In this work, the model is used to predict changes in the frameshift efficiency due to mutations in the viral mRNA sequence or variations in relative tRNA abundances. The model is sensitive to the size of the translating ribosome and to assumptions about the unfolding pathway of the stimulatory structure. As knowledge in the field of RNA structure prediction grows, that knowledge can be incorporated into the model developed here to make improved predictions. The single-ribosome translation model has been extended to polysomes by including initiation and termination rates and an exclusion principle, and allowing the stimulatory structure to refold on an appropriate timescale. The predicted frameshift efficiency for a given mRNA can be tuned by varying the ribosome density on the mRNA. This finding affects the interpretation of frameshift efficiencies measured in the lab. In the parameter regime where translation is initiation-limited, the frameshift efficiency also depends on the structure refolding rate, which determines the availability of the downstream structure for stimulating –1 frameshifts. Furthermore, there is a trade-off between frameshift efficiency and protein synthesis rate.

Small robust affinity proteins have shown great potential for use in therapy, in vivo diagnostics, and various biotechnological applications. However, the affinity proteins often need to be modified or functionalized to be successful in many of these applications. The use of chemical synthesis for the production of the proteins can allow for site-directed functionalization not achievable by recombinant routes, including incorporation of unnatural building blocks. This thesis focuses on chemical engineering of Affibody molecules and an albumin binding domain (ABD), which both are three-helix bundle proteins of 58 and 46 amino acids, respectively, possible to synthesize using solid phase peptide synthesis (SPPS). In the first project, an alternative synthetic route for Affibody molecules using a fragment condensation approach was investigated. This was achieved by using native chemical ligation (NCL) for the condensation reaction, yielding a native peptide bond at the site of ligation. The constant third helix of Affibody molecules enables a combinatorial approach for the preparation of a panel of different Affibody molecules, demonstrated by the synthesis of three different Affibody molecules using the same helix 3 (paper I). In the next two projects, an Affibody molecule targeting the amyloid-beta peptide, involved in Alzheimer’s disease, was engineered. Initially the N-terminus of the Affibody molecule was shortened resulting in a considerably higher synthetic yield and higher binding affinity to the target peptide (paper II). This improved variant of the Affibody molecule was then further engineered in the next project, where a fluorescently silent variant was developed and successfully used as a tool to lock the amyloid-beta peptide in a β-hairpin conformation during studies of copper binding using fluorescence spectroscopy (paper III). In the last two projects, synthetic variants of ABD, interesting for use as in vivo half-life extending partners to therapeutic proteins, were engineered. In the first project the possibility to covalently link a bioactive peptide, GLP-1, to the domain was investigated. This was achieved by site-specific thioether bridge-mediated cross-linking of the molecules via a polyethylene glycol (PEG)-based spacer. The conjugate showed retained high binding affinity to human serum albumin (HSA) and a biological activity comparable to a reference GLP-1 peptide (paper IV). In the last project, the possibility to increase the proteolytic stability of ABD through intramolecular cross-linking, to facilitate its use in e.g. oral drug delivery applications, was investigated. A tethered variant of ABD showed increased thermal stability and a considerably higher proteolytic stability towards pepsin, trypsin and chymotrypsin, three important proteases found in the gastrointestinal (GI) tract (paper V). Taken together, the work presented in this thesis illustrates the potential of using chemical synthesis approaches in protein engineering. / <p>QC 20140207</p>

<p>RNA localization provides the blueprint for compartmentalized protein synthesis in eukaryotic cells. Current paradigms indicate that RNAs encoding secretory and membrane proteins are recruited to the endoplasmic reticulum (ER), via positive selection of a `signal peptide' tag encoded in the protein. Thus RNA sorting to the ER follows protein sorting and the RNA is considered a passive player. However, RNAs have been shown to access the ER independent of the signal peptide and display a wide range of affinities to the ER that does not correlate with signal peptide strength. How and why mRNAs localize to the ER to varying extents and whether such localization serves a purpose besides protein sorting is poorly understood. To establish the cause and consequence of RNA binding to the ER membrane, I pose three primary questions: 1. How are mRNAs targeted to the ER? 2. Once targeted, how are mRNAs anchored to the ER membrane? 3. Are ER localized mRNAs subject to transcript-specific regulation? </p><p>I address cytosolic mRNA targeting to the ER by comparing the partitioning profiles of cytosolic/nuclear protein-encoding mRNA population (mRNACyto) to that of mRNAs encoding a signal peptide (mRNAER). I show that, at a population level, mRNACyto display a mean ER enrichment that is proportional to the amount of ER-bound ribosomes. Thus, I propose that targeting of mRNACyto to the ER is stochastic and over time, the specific interactions engaged by an individual mRNACyto with the ER determines its steady state partitioning profile between the cytoplasm and the ER. </p><p>To address the modes of direct binding of mRNA to the ER, I examined the association of various RNA populations with the ER after disrupting membrane-bound ribosome's interaction with its ER receptor. mRNACyto and most of mRNAs encoding secretory proteins (mRNACargo) are released upon disruption of ribosome-receptor interactions, indicating no direct mRNA-ER interactions. However, the population of mRNAs that encode resident proteins of the endomembrane organelles such as the ER, lysosome, endosome and the Golgi apparatus (mRNARes) maintain their association with the ER despite the disruption of ribosome-receptor interactions. These results indicate direct binding of mRNARes to the ER, further suggesting that the function of the encoded proteins dictates the mode of association of corresponding mRNA with the ER. </p><p>To uncover the mode of mRNARes binding directly to ER, I performed differential proteomic analysis of cytosolic and membrane bound RNA-protein complexes, which revealed a network of RNA binding proteins that interact uniquely with the ER-anchored mRNAs. The anchoring of endomembrane resident protein-encoding RNAs to the ER through these RNA binding proteins may reflect an imprinting of the ER with the information necessary for the continued biogenesis of the endomembrane organelle system even in situations where translation-dependent ER targeting of an mRNA is compromised. </p><p>Finally, I address whether ER-bound mRNAs can be regulated differentially by comparing the fates of two signal peptide-encoding RNAs, B2M and GRP94, during the unfolded protein response (UPR). I show that in response to ER stress, GRP94 mRNA, but not B2M, relocates to stress-induced RNA granules, thus escaping an RNA decay program that operates at the ER membrane during the UPR. Hence, I propose that the mode of RNA association to the ER is subject to regulation and influences the fate of RNAs during cellular stress. Thus, by demonstrating diverse modes of mRNA localization to the ER and differential regulation of ER bound mRNAs during cellular stress, my work has helped establish an emerging role for the ER as a post-transcriptional gene regulatory platform.</p> / Dissertation