Enzymes are attractive catalysts in organic synthesis since they are efficient, selective and environmentally friendly. A large number of enzyme-catalyzed transformations have been described in the literature. If no natural enzyme can carry out a desirable reaction, one possibility is to modify an existing enzyme by protein engineering and thereby obtain a catalyst with the desired properties. In this thesis, the development of enantioselective enzymes and their use in synthetic applications is described.  In the first part of this thesis, enantioselective variants of Candida antarctica lipase A (CALA) towards α-substituted p-nitrophenyl esters were developed by directed evolution. A highly selective variant of CALA towards p-nitrophenyl 2-phenylpropanoate was developed by pairwise randomization of amino acid residues close to the active site. The E value of this variant was 276 compared to 3 for the wild type. An approach where nine residues were altered simultaneously was used to discover another highly enantioselective CALA variant (E = 100) towards an ibuprofen ester. The sterical demands of this substrate made it necessary to vary several residues at the same time in order to reach a variant with improved properties. In the second part of the thesis, a designed variant of Candida antarctica lipase B (CALB) was employed in kinetic resolution (KR) and dynamic kinetic resolution (DKR) of secondary alcohols. The designed CALB variant (W104A) accepts larger substrates compared to the wild type, and by the application of CALB W104A, the scope of these resolutions was extended. First, a DKR of phenylalkanols was developed using CALB W104A. An enzymatic resolution was combined with in situ racemization of the substrate, to yield the products in up to 97% ee. Secondly, the KR of diarylmethanols with CALB W104A was developed. By the use of diarylmethanols with two different aryl groups, highly enantioselective transformations were achieved. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows:<strong>  </strong>Paper 5: Submitted.

Engineered affinity proteins have together with naturally derived antibodies becomeindispensable tools in many areas of life-science and with the increasing number ofapplications, the need for high-throughput methods for generation of such different affinityproteins is evident. Today, combinatorial protein engineering is the most successful strategy toisolate novel non-immunoglobulin affinity proteins. In this approach, generally termed directedevolution, high-complexity combinatorial libraries are created from which affinity proteins areisolated using an appropriate selection method, thus circumventing the need for detailedknowledge of the protein structure or the binding mechanism, often necessary in more rationalapproaches. Since the introduction of the phage display technology that pioneered the field ofcombinatorial engineering, several alternative selection systems have been developed for thispurpose.This thesis describes the development of a novel selection system based onstaphylococcal surface display and its implementation in directed evolution approaches. In thefirst study, the transformation efficiency to the gram-positive bacteria Staphylococcus carnosus wassuccessfully improved around 10,000-fold to a level that would allow cell surface display ofcomplex combinatorial protein libraries. In two separate studies, the staphylococcal displaysystem was investigated for the applicability in both de novo selection and affinity maturation ofaffibody molecules. First, using a pre-selection strategy with one round of phage display, ahigh-complexity affibody library was displayed on staphylococcal cells. Using fluorescenceactivatedcell sorting, binders with sub-nanomolar affinity to tumor necrosis factor-alpha(TNF-α) were isolated. Second, a combined approach using phage display for de novo selectionof first-generation affibody binders and staphylococcal display in a subsequent affinitymaturation selection was applied to generate binders with low nanomolar affinity to the humanepidermal growth factor receptor-3 (ErbB3). Moreover, in an additional study, thestaphylococcal surface display system was improved by the introduction of a protease 3Ccleavage sequence in the displayed fusion products in order to facilitate straightforwardproduction of soluble proteins for further downstream characterization.Altogether, the presented studies demonstrate that the staphylococcal selection systemindeed is a powerful tool for selection and characterization of novel affinity proteins and couldbecome an attractive alternative to existing selection techniques. / <p>QC 20100726</p>

This thesis describes generation and characterization of Affibody molecules with future applications in proteomics research, protein structure determinations, therapeutic treatment of disease and medical imaging for in vivo diagnostics. Affibody molecules are engineered affinity proteins developed by combinatorial protein engineering from the 58-residue protein A-derived Z domain scaffold. Novel Affibody molecules targeting human proteins were selected from a combinatorial library using phage display technology. In the first two investigations, an Affibody molecule specifically targeting the high abundant human serum protein transferrin was generated. The intended future use of this Affibody ligand would be as capture ligand for depletion of transferrin from human samples in proteomics analysis. Strong and highly specific transferrin binding of the selected Affibody molecule was demonstrated by biosensor technology, dot blot analysis and affinity chromatography. Efficient Affibody-mediated depletion of transferrin in human plasma and cerebrospinal fluid (CSF) was demonstrated in combination with IgG and HSA removal. Furthermore, depletion of five high abundant proteins including transferrin from human CSF gave enhanced identification of proteins in a shotgun proteomics analysis. Two studies involved the selection and characterization of Affibody molecules recognizing Alzheimer’s amyloid beta (Abeta) peptides. Future prospect for the affinity ligands would primarily be for therapeutic applications in treatment of Alzheimer’s disease. The developed A-binding Affibody molecules were found to specifically bind to non-aggregated forms of Abeta and to be capable of efficiently and selectively capture Abeta peptides from spiked human serum. Interestingly, the Abeta-binding Affibody ligands were found to bind much better to Abeta as dimeric constructs, and with impressive affinity as cysteine-bridged dimers (KD~17 nM). NMR spectroscopy studies revealed that the original helix one, of the two Affibody molecules moieties of the cysteine-bridged dimers, was unfolded upon binding, forming intermolecular β-sheets that stabilized the Abeta peptide, enabling a high resolution structure of the peptide. Furthermore, the Abeta-binding Affibody molecules were found to inhibit Abeta fibrillation in vitro. In the last study, Affibody molecules directed to the interleukin 2 (IL-2) receptor alpha (CD25) were generated. CD25-binding Affibody molecules could potentially have a future use in medical imaging of inflammation, and possibly in therapeutic treatment of disease conditions with CD25 overexpression. The selected Affibody molecules were demonstrated to bind specifically to human CD25 with an apparent affinity of 130-240 nM. Moreover, the CD25-targeting Affibody molecules were found to have overlapping binding sites with the natural ligand IL-2 and an IL-2 blocking monoclonal antibody. Furthermore, the Affibody molecules demonstrated selective binding to CD25 expressing cells. / QC 20100729

Tumor targeting and molecular imaging of protein markers specific for or overexpressed in tumors can add useful information in deciding upon treatment and assessing the response to treatment for a cancer patient. The epidermal growth factor receptor (EGFR) is one such tumor-associated receptor, which expression is abnormal or upregulated in various cancers and associated with a poor patient prognosis. It is therefore considered a good target for imaging and therapy. Monoclonal antibodies and recently also antibody fragments have been investigated for in vivo medical applications, like therapy and imaging. In molecular imaging a small sized targeting agent is favorable to give high contrast and therefore, antibody fragments and lately also small affinity proteins based on a scaffold structure constitute promising alternatives to monoclonal antibodies. Affbody molecules are such affinity proteins that are developed by combinatorial protein engineering of the 58 amino acid residue Z-domain scaffold, derived from protein A. In this thesis, novel Affibody molecules specific for the EGFR have been selected from a combinatorial library using phage display technology. Affibody molecules with moderate high affinity demonstrated specific binding to native EGFR on the EGFR-expressing epithelial carcinoma A431 cell line. Further cellular assays showed that the EGFR-binding Affibody molecules could be labeled with radiohalogens or radiometals with preserved specific binding to EGFR-expressing cells. In vitro, the Affibody molecule demonstrated a high uptake and good retention to EGFR-expressing cells and was found to internalize. Furthermore, successful imaging of tumors in tumor-bearing mice was demonstrated. Low nanomolar or subnanomolar affinities are considered to be desired for successful molecular imaging and a directed evolution to increase the affinity was thus performed. This resulted in an approximately 30-fold improvement in affinity, yielding EGFR-binding Affibody molecules with KD´s in the 5-10 nM range, and successful targeting of A431 tumors in tumor-bearing mice. To find a suitable format and labeling, monomeric and dimeric forms of one affinity matured binder were labeled with 125I and 111In. The radiometal-labeled monomeric construct, 111In-labeled-ZEGFR:1907, was found to provide the best tumor-to-organ ratio due to good tumor localization and tumor retention. The tumor-to-blood ratio, which is often used as a measure of contrast, was 31±8 at 24 h post injection and the tumor was clearly visualized by gamma-camera imaging. Altogether, the EGFR-binding Affibody molecule is considered a promising candidate for further development of tumor imaging tracers for EGFR-expressing tumors and metastases. This could simplify the stratification of patients for treatment and the assessment of the response of treatment in patients. / QC 20100723

Even though our understanding of mechanisms such as protein folding and molecular recognition is relatively poor, antibodies and alternative affinity proteins with entirely novel functions are today generated in a routine manner. The reason for this success is an engineering approach generally known as directed evolution. Directed evolution has provided researchers with a tool for circumventing our limited knowledge and hence the possibility to create novel molecules that by no means could have been designed today. The approach is based on construction of high-complexity combinatorial libraries from which protein variants with desired properties can be selected. Engineered proteins are already indispensable tools in nearly all areas of life science and the recent advent of mainly monoclonal antibodies as therapeutic agents has directed even more attention to the field of combinatorial protein engineering. In this thesis, I present the underlying research efforts of six original papers. The overall objective of the studies has been to develop and investigate a new staphylococcal surface display method for protein engineering and protein characterization. The technology is based on display of recombinant proteins on surface of the Gram-positive bacteria Staphylococcus carnosus. In two initial studies, two key issues were addressed in order to improve the protein engineering method in regard to affinity discrimination ability and transformation efficiency. The successful results enabled investigation of the staphylococcal display system for de novo generation of affibody molecules from large combinatorial libraries. In this study, a high-complexity protein library was for the first time displayed on surface of Gram-positive bacteria and by means of fluorescence-activated cell sorting, specific affinity proteins for tumor necrosis factor-alpha were isolated. Moreover, in following papers, the staphylococcal display method was further improved and investigated for affinity determination, soluble protein production and epitope mapping purposes in order to facilitate downstream characterizations of generated affinity proteins. Taken together, in these studies we have demonstrated that the staphylococcal display system is a powerful alternative to existing technologies for protein engineering and protein characterization. / QC 20100809

Enzymes are gaining increasing importance as catalysts for selective transformations in organic synthetic chemistry. The engineering and design of enzymes is a developing, growing research field that is employed in biocatalysis. In the present thesis, combinatorial protein engineering methods are applied for the development of Candida antarctica lipase A (CALA) variants with broader substrate scope and increased enantioselectivity. Initially, the structure of CALA was deduced by manual modelling and later the structure was established by X-ray crystallography. The elucidation of the structure of CALA revealed several biocatalytically interesting features. With the knowledge derived from the enzyme structure, enzyme variants were produced via iterative saturation mutagenesis (ISM), a powerful protein engineering approach. Several of these variants were highly active and enantioselective towards bulky esters. Furthermore, an extensively combinatorial protein engineering approach was developed and investigated. A CALA variant with a spacious substrate binding pocket that can accommodate an unusually bulky substrate, an ester derivate of the non-steroidal anti-inflammatory drug (S)-ibuprofen, was obtained with this approach. / At the time of the doctoral defence the following paper was unpublished and had a status as follows: Paper nr. 5: Manuscript

(S)-3-O-Geranylgeranylglyceryl Phosphate Synthase (GGGPS) is a TIM (βα)8 barrel protein found in Archaea and the enzyme catalyzing the first step in the biosynthesis of archaeal membrane lipids. The TIM (βα)8 barrel protein fold is thought to have evolved by duplication and fusion of (βα)4 half barrels. We propose that the GGGPS has also evolved from (βα)4 half barrels. One way to test this hypothesis is to generate putative half-barrels experimentally. GGGPS from Archaeaglobus fulgidus, is a dimer of (βα)8 barrels. Thus, before constructing half barrels, a stable monomer is needed to be engineered. Introducing three substitutions into the dimer interface formed the GGGPS monomer. AUC showed ~50 % of the protein is in the monomeric state. CD experiments confirmed that the engineered protein was properly folded but had decreased thermal stability. In an enzymatic assay, the monomeric GGGPS protein proved as active as the WT protein on a subunit basis.

(S)-3-O-Geranylgeranylglyceryl Phosphate Synthase (GGGPS) is a TIM (βα)8 barrel protein found in Archaea and the enzyme catalyzing the first step in the biosynthesis of archaeal membrane lipids. The TIM (βα)8 barrel protein fold is thought to have evolved by duplication and fusion of (βα)4 half barrels. We propose that the GGGPS has also evolved from (βα)4 half barrels. One way to test this hypothesis is to generate putative half-barrels experimentally. GGGPS from Archaeaglobus fulgidus, is a dimer of (βα)8 barrels. Thus, before constructing half barrels, a stable monomer is needed to be engineered. Introducing three substitutions into the dimer interface formed the GGGPS monomer. AUC showed ~50 % of the protein is in the monomeric state. CD experiments confirmed that the engineered protein was properly folded but had decreased thermal stability. In an enzymatic assay, the monomeric GGGPS protein proved as active as the WT protein on a subunit basis.

Ca2+, a signal for death and life, is closely involved in the regulation of numerous important cellular events. Ca2+ carries out its function through its binding to Ca2+-receptors or Ca2+-binding proteins. The EF-hand protein, with a helix-loop-helix Ca2+-binding motif, constitutes one of the largest protein families. To facilitate our understanding of the role of Ca2+ in biological systems (denoted as calciomics) using genomic information, an improved pattern search method (http://www.chemistry.gsu.edu/faculty/Yang/Calciomics.htm) for the identification of EF-hand and EF-like Ca2+-binding proteins was developed. This fast and robust method allows us to analyze putative EF-hand proteins at the genome-wide level and further visualize the evolutionary scenario of the EF-hand protein family. This prediction method further enables us to locate a putative viral EF-hand Ca2+-binding motif within the rubella virus nonstructural protease that cleaves the nonstructural protein precursor into two active replicase components. A novel grafting approach has been used to probe the metal-binding properties of this motif by engineering the predicted 12-residue Ca2+-coordinating loop into a non-Ca2+-binding scaffold protein, CD2 domain 1. Structural and conformational studies were further performed on a purified, bacterially-expressed NS protease minimal metal-binding domain spanning the Zn2+- and EF-hand Ca2+-binding motif. It was revealed that Ca2+ binding induced local conformational changes and increased thermal stability. Furthermore, functional studies were carried out using RUB infectious cDNA clone and replicon constructs. Our studies have shown that the Ca2+ binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions. In addition, we have predicted and characterized a calmodulin-binding domain in the gap junction proteins connexin43 and connexin44. Peptides encompassing the CaM binding motifs were synthesized and their ability to bind CaM was determined using various biophysical approaches. Transient expression in HeLa cells of two mutant Cx43-EYFP constructs without the putative CaM-binding site eliminated the Ca2+-dependent inhibition of gap junction permeability. These results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 and Cx44 in a Ca2+-dependent manner, providing a molecular basis for the well-characterized Ca2+-dependent inhibition of Cx43-containing gap junctions.

Ca2+, both as a first and a second messenger, is closely involved in the modulation and regulation of numerous important cellular events, such as cell proliferation, differentiation and cell death. Fine-tuned Ca2+ signaling is achieved by its reversible or irreversible binding to a repertoire of Ca2+ signaling molecules. Among them, the extracellular calcium sensing receptor (CaSR) senses Ca2+ concentration ([Ca2+]o) in the milieu outside of cells where Ca2+ serves as a first messenger. An array of naturally-occurring mutations in CaSR has been found in patients with inherited disorders of Ca2+ homeostasis, leading to abnormal intracellular responses toward [Ca2+]o. In the present study, we have computationally predicted and experimentally characterized the metal-binding properties of five Ca2+-binding sites within CaSR and the accompanying metal--induced conformational changes by using two complementary methods-the grafting approach and the subdomain approach. Based on our results, a model has been proposed to explain the distinct CaSR-mediated responses toward abnormally ¡°high¡± or ¡°low¡± extracellular Ca2+ levels. In addition, we predicted and verified the interaction between CaSR with the most ubiquitously expressed four EF-hand-containing intracellular Ca2+ sensor protein, calmodulin (CaM). Our results demonstrate that the C-terminal CaM-binding domain of the CaSR is essential for proper intracellular Ca2+ response to external signals. Furthermore, we have applied the grafting approach to study the metal-binding properties and oligomeric state of the single EF-hand containing protein, STIM1. Our studies confirmed that the single EF-hand motif in STIM1, which resides in an equilibratium between its monomeric and dimeric forms, was capable of binding Ca2+ with a dissociation constant comparable to the ER Ca2+ concentration, suggesting it could function as a ER Ca2+ sensor responsible for sensing the Ca2+ filling state of ER.