| 1. |
- Löfdahl, Per-Åke, 1959-
(författare)
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On bacterial formats in protein library technology
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Millions of years of evolution have resulted in an immense number of different proteins, which participate in virtually every process within cells and thus are of utmost importance for allknown forms of life. In addition, there are several examples of natural proteins which have found use in applications outside their natural environment, such as the use of enzymes infood industry and washing powders or the use of antibodies in diagnostic, bioseparation or therapeutic applications. To improve the performance of proteins in such applications, anumber of techniques, all collectively referred to as ‘protein engineering’, are performed in thelaboratory.Traditionally, methods involving ‘rational design’, where a few alterations are introduced atspecific protein locations to hopefully result in expected improvements have been applied.However, the use of more recent techniques involving a simultaneous construction of a large number of candidate variants (protein libraries) by various diversification principles, fromwhich rare clones showing enhanced properties can be isolated have contributed greatly to thefield of protein engineering.In the present thesis, different protein traits of biotechnological importance have beenaddressed for improvements by the use of such methods, in which there is a crucial need tomaintain a clonal link between the genotype and the phenotype to allow an identification of protein library members isolated by virtue of their functional properties. In all protein library investigations included in this thesis this coupling has been obtained by Escherichia coli bacterialcell-membrane compartmental confinement.In a first study, a combination of error prone PCR and gene-shuffling was applied to the Tobacco Etch Virus (TEV)-protease gene in order to produce collections from which genesencoding variants showing an enhanced soluble expression of the enzyme frequently used inbiotechnology to cleave fusion proteins were identified. Using Green Fluorescence Protein(GFP)-based cell fluorescence analysis, a clone with a five-fold increase in the yield of solubly produced protein was successfully isolated. In a second study, a novel and different GFPbased selection system, in addition also involving targeted in vivo protein degradation principles,was employed for investigations of the substrate sequence space of the same protease. In two additional studies, a selection system denoted Protein Fragment Complementation Assay(PCA), based on the affinity driven structural complementation of a genetically split β-lactamase enzyme was used to identify variants having desired target protein binding abilities,including both specificity and affinity. Using Darwinian principles concerning clonal growth advantages, affibody binding proteins showing sub-nanomolar dissociation constants to thehuman cytokine TNF-α were isolated. Taken together, these studies have shown that the bacterial format is very well suited for use in various aspects of protein library selection.
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| 2. |
- Myrhammar, Anders, 1987-
(författare)
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Investigations of chemical and enzymatic functionalization of affinity proteins
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- AbstractAffinity proteins are important reagents in research, diagnostics and therapeutic settings. The focus of this thesis has been on investigating different chemical and enzymatic strategies for engineering of affinity proteins to generate affinity reagents with improved or changed functionality. The modifications introduced in affibodies, representing a class of small, three-helix engineered scaffold proteins, and antibodies were selected and implemented through rational design, using a combination of solid phase peptide synthesis, genetic engineering and enzymatic conjugation, depending on the case.In a first study, thioether crosslinks were introduced between internally positioned lysines and cysteines of the human epidermal growth factor receptor (hEGFR)-targeting affibody ZEGFR:1907, to test the possibility to increase the proteolytic stability of the affibody scaffold. Three different variants of crosslinked affibodies were produced, containing one or two crosslinks. All three variants showed similar affinities to EFGR, and secondary structure contents, as the unmodified control protein. The crosslinked affibodies were challenged with the endopeptidases pepsin, found in the stomach, and trypsin and chymotrypsin, found in the gut. All affibodies showed improved stability towards at least one of the proteases, but the largest improvement was seen for the affibody harboring two crosslinks, which displayed the greatest stability in both assays.Improvement in proteolytic stability of affibodies was further explored. In another study a sortase A-catalyzed intramolecular head-to-tail conjugation of the dimeric human epidermal growth factor 2 (HER2)-targeting affibody (ZHER2:342)2 was performed. Analysis showed no change in α-helicity for the cyclic dimer compared to the linear control, and a slight increase in melting temperature. Interestingly, in contrast to the linear variant, the cyclic dimer showed no signs of proteolytic degradation after 60 min exposure to the exopeptidase carboxypeptidase A.The ability to change protein functionality by chemical modification was explored in two studies. The immunoglobulin-binding Z domain, from which the affibody scaffold is derived, was used as a model protein in one study, where light-induced affinity modulation was investigated. An azobenzene switch that isomerizes from a trans to a cis state was introduced end-to-end to one of the helices in three different designs of the Z domain. The conformational change induced by isomerization was hypothesized to be large enough to cause a loss in binding affinity in the conjugated affibody, which was tested in an affinity chromatography assay in which one of the affibodies captured to an IgG-sepharose column showed loss of affinity during illumination.Peptide nucleic acid (PNA) probes have previously successfully been used for selective hybridization between the primary, tumor-targeting agent and the secondary agent in a pretargeting set-up for in vivo tumor imaging or directed therapy. In a last study, a Z domain-PNA conjugate produced via sortase A-mediated conjugation was photoconjugated to a lactosaminated antibody for possible use as an in vivo clearing agent for clearance of excess of primary probes via an hepatic route. The clearing agent showed partial success in a mouse model but the concept needs further work.The work in this thesis shows the diverse possibilities available for changing the functionality of affinity proteins through chemical and enzymatic methods for different applications, and provides a framework for potential further improvement of both affibody and antibody functionality.
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| 3. |
- Zhang, Jie
(författare)
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Targeting Human Epidermal Growth Factor Receptors with Drug Conjugates Based on Affibody Molecules
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer is a major public health challenge and the second leading cause of death in the world, with millions of new cases being diagnosed each year. Traditional cancer treatments such as surgery, radiation therapy, and chemotherapy are many times effective, but may also cause damage to healthy cells, leading to side effects. Targeted therapy is a more precise and focused approach to cancer treatment, where the aim is to target the cancer cells while leaving the normal cells unaffected. It is particularly effective in cancers where specific molecular targets are known, such as the subset of breast cancer patients with HER2 over-expression or in the subset of patients with pancreatic cancer with HER3 over-expression. Antibody-drug conjugates (ADCs) are an important addition to tumor-targeted therapy, with twelve drugs approved for clinical use by the FDA. They utilize the high specificity of monoclonal antibodies conjugated with highly cytotoxic small molecules to enhance the accumulation of the drugs in the tumor, for highly specific and efficient killing. However, traditional ADCs may not be the optimal delivery format for the directed delivery of cytotoxic drugs. They are limited by their relatively large molecular weights, resulting in relatively low penetration of solid tumors. Recently, a novel type of drug conjugates, affibody-drug conjugates, has been described. These combined an engineered scaffold affinity protein, an affibody molecule, with an albumin binding domain (ABD) for half-life extension, to which a cytotoxic payload has been conjugated. Previous studies show that these novel drug conjugates have a potent and tumor-cell-specific cytotoxic effect. In the future, they may therefore become complementary or alternatives to current targeted cancer therapies. This thesis focuses on the optimization of affibody-drug conjugates targeting HER2 and HER3, members of the human epidermal growth factor receptor family. The thesis presents in vitro and in vivo preclinical data, showing the potential for further clinical development. In paper I, we investigated the influence of the drug-to-affibody ratio (DAR) on the pharmacokinetic profile of affibody-drug conjugates targeting HER2. Increasing the drug load resulted in an elevated delivery of the DM1 drug to the tumors; however, it also led to increased uptake by the liver. Further optimization of the molecular design is necessary to enable highly efficient delivery to tumors while minimizing the uptake in normal organs and tissues. In paper II, the effect of the length and composition of the linker between the HER2 targeting affibody molecule and the ABD was investigated. The use of a 12 amino acids linker reduced hepatic uptake compared with the use of a 5 amino acids linker. This finding offers an important insight into the influence of the linker on the properties of the affibody drug conjugates. In paper III, we investigated the influence of different cytotoxic payloads, as part of an affibody-drug conjugate targeting HER2, on binding properties, cytotoxicity, biodistribution, and anti-tumor effect. The combination of a potent cytotoxic effect in vitro, and a high tumor uptake in vivo, resulted in a superior anti-tumor effect for ZHER2- ABD-mcMMAF at lower doses compared to the previously investigated ZHER2-ABD- mcDM1. Importantly, it maintained a favorable toxicity profile with lower liver uptake compared to ZHER2-ABD-mcDM1. The affibody-drug conjugate ZHER2-ABD- mcMMAF holds great promise as a valuable agent for HER2-targeted cancer therapy. In paper IV, we generated a series of HER2-targeted affibody-drug conjugates fused with different PAS or XTEN polypeptides. We evaluated the ability of the XTEN and PAS polypeptides to extend the plasma half-life, and their influence on tumor uptake, and tissue biodistribution. We compared our new constructs with the previously developed construct, ZHER2-ABD-mcDM1, where an albumin binding domain was used for half-life extension. It was found that the ABD-fused affibody-drug conjugate demonstrated superior tumor uptake and tumor-to-normal-organ ratios compared to the PASylated and XTENylated affibody-drug conjugates. It is possible that ABD is better also for other cancer-targeting strategies where a high tumor uptake while maintaining comparable accumulation in normal tissues is desired. In paper V, we compared the binding properties and cytotoxic potential of a monovalent and a bivalent HER3-targeting affibody-drug conjugate. The biodistribution and therapeutic potential of the bivalent drug construct were evaluated. We found that the bivalent ZHER3-ABD-ZHER3- mcDM1 is a highly potent drug conjugate with favorable biodistribution and anti-tumor efficacy. These results suggest that ZHER3-ABD-ZHER3-mcDM1 holds promise for future clinical development as a potential therapeutic option for patients with HER3 over-expressing cancer. In summary, the potential for modification and optimization through the design of diverse components within HER2 and HER3-targeting affibody-drug conjugates significantly enhances therapeutic effectiveness, thereby encouraging prospective advancements in the development of targeted drug conjugates.
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| 4. |
- Tano, Hanna
(författare)
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PNA and affinity protein tools for selective tumor targeting of radiopharmaceuticals
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Targeted radiotherapy of cancer intends to selectively deliver cytotoxic radionuclides to tumor cells. Affinity proteins of various kinds are explored for this task, and depending on the affinity protein used, different challenges arise. Full-length antibodies are typically associated with long serum half-life, leading to high systemic toxicity, while smaller affinity ligands such as engineered scaffold proteins, antibody fragments or peptides, usually demonstrate high radioactive uptake in kidneys. The smallest affinity ligands furthermore suffer from low therapeutic efficacy due to their fast wash-out, thus demanding frequent administrations of the radio-conjugate to reach a therapeutic effect. These issues were addressed in this thesis, where small affinity ligands (an Affibody molecule, a single domain antibody fragment and a peptide) have been explored as targeting agents for the cancer targets HER2, CD38 and GRPR, respectively. The Affibody molecule and the single domain antibody fragment were used in a pretargeting setting where high selective hybridization are used as recognition tags between peptide nucleic acid (PNA) strands on the tumor targeting primary agent and the radiolabelled secondary agent. In papers I and II, different sets of PNA hybridization probes were evaluated, in vitro and in vivo. In paper I, we demonstrate that the shortest tested secondary PNA probe (the 9-mer HP16) had the most favourable biodistribution profile with high tumor uptake along with the lowest kidney uptake. In paper II, we produced a set of shorter primary PNA probes, aiming for simplified production, and new sets of even shorter secondary PNA probes. A secondary 8-mer was identified as suitable for testing in cell assays and in vivo together with HER2-binding Affibody-PNA conjugates with varying length of the primary PNA probe, in order to determine if the smaller hydrodynamic range would further improve the biodistribution properties. In paper III, the Affibody-mediated PNA-based pretargeting strategy was evaluated as a monotherapy and as a co-treatment strategy with trastuzumab, to treat mice bearing HER2-positive tumors. Mice treated with the co-treatment strategy had significantly longer survival compared to other groups. In paper IV, the feasibility of using the PNA pretargeting strategy in combination with another affinity protein (a single domain antibody fragment) was evaluated in a CD38-expressing cell line. In paper V, the GRPR-binding peptide RM26 was conjugated to an albumin-binding domain, with the aim to achieve a high tumor uptake over time. The RM26-ABD conjugate did demonstrate good tumor uptake over time. However, the conjugate also demonstrated high kidney uptake, limiting its use as a therapeutic construct. In conclusion, the work presented in this thesis shows strategies for selective tumor targeting of radiopharmaceuticals using affinity proteins and PNA-mediated pretargeting.
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| 5. |
- Dogan, Jakob, 1978-
(författare)
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Structural and thermodynamical basis for molecular recognition between engineered binding proteins
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The structural determination of interacting proteins, both as individual proteins and in their complex, complemented by thermodynamical studies are vital in order to gain in-depth insights of the phenomena leading to the highly selective protein-protein interactions characteristic of numerous life processes. This thesis describes an investigation of the structural and thermodynamical basis for molecular recognition in two different protein-protein complexes, formed between so-called affibody proteins and their respective targets. Affibody proteins are a class of engineered binding proteins, which can be functionally selected for binding to a given target protein from large collections (libraries) constructed via combinatorial engineering of 13 surface-located positions of the 58-residue three-helix bundle Z domain derived from Staphylococcal protein (SPA). In a first study, an affibody:target protein pair consisting of the ZSPA-1 affibody and the parental Z domain, with a dissociation constant (Kd) of approximately 1 µM, was investigated. ZSPA-1 was in its free state shown to display molten globule-like characteristics. The enthalpy change on binding between Z and ZSPA-1 as measured by isothermal titration calorimetry, was found to be a non-linear function of temperature. This nonlinearity was found to be due to the temperature dependent folded-unfolded equilibrium of ZSPA-1 upon binding to the Z domain and, the energetics of the unfolding equilibrium of the molten globule state of ZSPA-1 could be separated from the binding thermodynamics. Further dissection of the binding entropy revealed that a significant reduction in conformational entropy resulting from the stabilization of the molten globule state of ZSPA-1 upon complex formation could be a major reason for the moderate binding affinity. A second studied affibody:target complex (Kd ~ 0.1 µM) consisted of the ZTaq affibody protein originally selected for binding to Taq DNA polymerase and the anti-ZTaq affibody protein, selected for selective binding to the ZTaq affibody protein, thus constituting an "anti-idiotypic" affinity protein pair. The structure of the ZTaq:anti-ZTaq affibody complex as well as the free state structures of ZTaq and anti-ZTaq were determined using NMR spectroscopy. Both ZTaq and anti-ZTaq are well defined three helix bundles in their free state and do not display the same molten globule-like behaviour of ZSPA-1. The interaction surface was found to involve all of the varied positions in helices 1 and 2 of the anti-ZTaq, the majority of the corresponding side chains in ZTaq, and also several non-mutated residues. The total buried surface area was determined to about 1670 Å2 which is well inside the range of what is typical for many protein-protein complexes, including antibody:antigen complexes. Structural rearrangements, primarily at the side chain level, were observed to take place upon binding. There are similarities between the ZTaq:anti-ZTaq and the Z:ZSPA-1 structure, for instance, the binding interface area in both complexes has a large fraction of non-polar content, the buried surface area is of similar size, and certain residues have the same positioning. However, the relative orientation between the subunits in ZTaq:anti-ZTaq is markedly different from that observed in Z:ZSPA-1. The thermodynamics of ZTaq:anti-ZTaq association were investigated by isothermal titration calorimetry. A dissection of the entropic contributions showed that a large and favourable desolvation entropy of non-polar surface is associated with the binding reaction which is in good agreement with hydrophobic nature of the binding interface, but as in the case for the Z:ZSPA-1 complex a significant loss in conformational entropy opposes complex formation. A comparison with complexes involving affibody proteins or SPA domains suggests that affibody proteins inherit intrinsic binding properties from the original SPA surface. The structural and biophysical data suggest that although extensive mutations are carried out in the Z domain to obtain affibody proteins, this does not necessarily affect the structural integrity or lead to a significant destabilization.
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| 6. |
- Grimm, Sebastian, 1980-
(författare)
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Ribosome display for selection and evolution of affibody molecules
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Affinity proteins are invaluable tools in biotechnological and medical applications. This thesis is about combinatorial protein engineering principles for the generation of novel affinity proteins to purify mouse immunoglobulin, detect a potential cancer marker protein or inhibit a cell proliferation pathway. In a first study, ribosome display was for the first time applied to the selection of so-called affibody molecules, including the design of a ribosome display gene cassette, initial test enrichment experiments and the selection of binders against murine IgG1. One of the selected binders (ZMAB25) showed a highly selective binding profile to murine IgG1, which was exploited in the recovery of two different mouse monoclonal IgG1 antibodies from a bovine immunoglobulin-containing background. Ribosome display was further applied to the selection of affibody molecules binding to SATB1, a suggested marker protein for metastasizing adenocarcinoma. The study also included the selection of VHH antibody fragments from a naïve gene repertoire displayed on phage. Binders from both classes of protein scaffolds could be isolated that selectively recognized SATB1 but not its close homologue SATB2, and were used to detect endogenous SATB1 in Jurkat cells by immunofluorescence microscopy. The well-established phage display technology was used to select affibody molecules binding to H-Ras and Raf-1, both involved in the mitogen-activated protein kinase (MAPK) pathway and playing a central role in the control of cell proliferation, survival and differentiation. An isolated affibody molecule denoted ZRAF322 was found to selectively bind to Raf-1 and inhibit the interaction between H-Ras and Raf-1 in vitro. In a continued effort, ribosome display was applied to the affinity maturation of the ZRAF322 variant in a novel approach, based on repetitive cycles of diversification by error-prone PCR of the entire affibody gene and ribosome display selection, mimicking the principles of natural evolution. The method involved a monitoring of the progress of evolution and variants of ZRAF322 with 13- to 26-fold improved affinities were obtained, that contained different combinations of single or double amino acid substitutions in either previously randomized or framework positions. Implications of the substitutions for binder stability and selectivity were also investigated, showing that a higher affinity could be associated with a lower thermal melting point and that affinity-improved variants showed uncompromised binding selectivity to the hRaf-1 target.
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| 7. |
- Gustafsdottir, Sigrun Margret, 1978-
(författare)
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Application of proximity Ligation for Detection of Proteins, Biomolecular Interactions, and Single Copies of Pathogens
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proximity ligation is a recently established technique that can provide answers to questions about the concentration, localization, interactions, modifications and functions of proteins. The method enables sensitive protein measurements with a detection limit in the low femtomolar range in complex biological samples. In proximity ligation, the challenge of detecting specific proteins is converted to the analysis of specific DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins, and to form amplifiable tag sequences upon ligation when brought in proximity. Protocols for the conversion of monoclonal or polyclonal antibodies into proximity probes through the attachment of oligonucleotide sequences are described in the thesis. In addition, the thesis describes the adaptation of the proximity ligation technology for detection of microbial pathogens, analysis of interactions between proteins and nucleic acids, and of inhibition of receptor-ligand interactions. Nucleic acid amplification allows specific detection of pathogens with single-copy sensitivity. There are many circumstances, however, when analysis of pathogen surface antigens or the antibody response can provide increased diagnostic value. Proximity ligation reactions were used to measure numbers of virus and bacteria by detection of viral or bacterial surface proteins. Detection sensitivities similar to those of nuclear acid-based detection reactions were achieved directly in infected samples for a parvovirus and for an intracellular bacterium. Biological processes are orchestrated by interactions of proteins with molecules in their environment, and investigations of interactions between proteins and other biomolecules are thus of great importance. Protocols were established for very specific and sensitive homogeneous-phase analysis of interactions between proteins and specific nucleic acid sequences. Finally, the proximity ligation mechanism was used to monitor interactions between VEGF-A and two of its receptors, VEGFR-1 and VEGFR-2, and to characterize the effects of agents disrupting this interaction.
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| 8. |
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| 9. |
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| 10. |
- Wahlberg, Elisabet, 1963-
(författare)
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Structure determination and thermodynamic stabilization of an engineered protein-protein complex
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The interaction between two 6 kDa proteins has been investigated. The studied complex of micromolar affinity (Kd) consists of the Z domain derived from staphylococcal protein A and the related protein ZSPA-1, belonging to a group of binding proteins denoted affibody molecules generated via combinatorial engineering of the Z domain. Affibody-target protein complexes are good model systems for structural and thermodynamic studies of protein-protein interactions. With the Z:ZSPA-1 pair as a starting point, we determined the solution structure of the complex and carried out a preliminary characterization of ZSPA-1. We found that the complex contains a rather large (ca. 1600 Å2) interaction interface with tight steric and polar/nonpolar complementarity. The structure of ZSPA-1 in the complex is well-ordered in a conformation that is very similar to that of the Z domain. However, the conformation of the free ZSPA-1 is best characterized by comparisons with protein molten globules. It shows a reduced secondary structure content, aggregation propensity, poor thermal stability, and binds the hydrophobic dye ANS. This molten globule state of ZSPA-1 is the native state in the absence of the Z domain, and the ordered state is only adopted following a stabilization that occurs upon binding. A more extensive characterization of ZSPA-1 suggested that the average topology of the Z domain is retained in the molten globule state but that it is represented by a multitude of conformations. Furthermore, the molten globule state is only marginally stable, and a significant fraction of ZSPA-1 exists in a completely unfolded state at room temperature. A complete thermodynamic characterization of the Z:ZSPA-1 pair suggests that the stabilization of the molten globule state to an ordered three helix structure in the complex is associated with a significant conformational entropy penalty that might influence the binding affinity negatively and result in an intermediate-affinity (µM) binding protein. This can be compared to a dissociation constant of 20-70 nM for the complex Z:Fc of IgG where Z uses the same binding surface as in Z:ZSPA-1. Structure analyses of Z in the free and bound state reveal an induced fit response upon complex formation with ZSPA-1 where a conformational change of several side chains in the binding surface increases the accessible surface area with almost 400 Å2 i.e. almost half of the total interaction surface in the complex. Two cysteine residues were introduced at specific positions in ZSPA-1 for five mutants in order to stabilize the conformation of ZSPA-1 by disulfide bridge formation. The mutants were thermodynamically characterized and the binding affinity of one mutant showed an improvement by more than a factor of ten. The improvement of the introduced cysteine bridge correlates with an increase in binding enthalpy rather than with entropy. Further analysis of the binding entropy suggests that the conformational entropy change in fact is reduced but its favorable contribution is opposed by a less favorable desolvation enthalpy change. These studies illustrate the structural and thermodynamic complexity of protein-protein interactions, but also that this complexity can be dissected and understood. In this study, a comprehensive characterization of the ZSPA-1 affibody has gained insight into the intricate mechanisms involved in complex formation. These theories were supported by the design of a ZSPA-1 mutant with improved binding affinity.
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