Lotta Tegler

Powerful protein binders from designed polypeptides and small organic molecules: a general concept for protein recognition

In this thesis, the interactions between different proteins and small ligands were characterized by surface plasmon resonance spectroscopy (SPR) and fluorescence resonance energy transfer (FRET) based assays. For the C-reactive protein (CRP), a new type of artificial binder was identified which allows designing diagnostic assays superior to commonly used standard assays. Furthermore, an interaction study with the endogenous ligand phosphocholine revealed the importance of the avidity of pentameric CRP for the distinction of different types of lipid membranes. The interaction study with calcium showed how SPR based assays can be used to study ion-protein interactions despite the low atomic weight of ions. The transmembrane protease BACE1, an important drug target for Alzheimer’s disease, was immobilized to an SPR biosensor surface and embedded into a lipid membrane. An interaction study with a set of known BACE1 inhibitors showed that the transmembrane region has only minor effects on the interactions. Furthermore the pH-dependencies of the interactions were investigated and revealed new important conclusions for inhibitor design. Computer aided modelling showed that the protonation state of the aspartic dyad is dependent on the interacting inhibitor which offers new perspectives for in silico screenings.The SPR assay developed for BACE1 was adapted to a more complex membrane protein, the pentameric β3 GABAA receptor. The assay allowed the pharmacological characterisation for histaminergic and GABAergic ligands and gave further evidence for cross-talk between the two signal transduction pathways. This study shows that the immobilisation method used for BACE1 and the s3 GABAA receptor has the potential to become a standard method for handling membrane proteins. The identification of new drug leads from natural sources is a common strategy for drug discovery. A combination of SPR and FRET based activity assays were explored to increase the efficiency of this process. For HIV-1 protease, secreted aspartic protease (SAP) 1, 2 and 3 extracts from a marine vertebrate were identified containing potent inhibitors which interacted with the active site of the enzymes.The studies in this thesis show that the investigation of protein interactions is crucial for understanding protein functions and can help to develop novel drugs for the treatment of different diseases.

Polypeptide conjugate binders that discriminate between two isoforms of human carbonic anhydrase in human blood.

Two binder candidates 4‐C37L34‐B and 3‐C15L8‐B from a 16‐membered set of 42‐residue polypeptide conjugates designed to bind human carbonic anhydrase II (HCAII), were shown to bind HCAII with high affinity in a fluorescence‐based screening assay. Two carbonic anhydrase isoforms with 60 % homology exist in human blood with HCAI being present in five‐ to sevenfold excess over HCAII. The ability of the binders to discriminate between HCAI and HCAII was evaluated with regard to what selectivity could be achieved by the conjugation of polypeptides from a 16‐membered set to a small organic molecule that binds both isoforms with similar affinities. The polypeptide conjugate 4‐C37L34‐B bound HCAII with a KD of 17 nM and HCAI with a KD of 470 nM, that is, with a 30‐fold difference in affinity. The corresponding dissociation constants for the complexes formed from 3‐C15L8‐B and the two carbonic anhydrases were 60 and 390 nM, respectively. This demonstration of selectivity between two very similar proteins is striking in view of the fact that the molecular weight of each one of the conjugate molecules is little more than 5000, the fold is unordered, and the polypeptide sequences were designed de novo and have no prior relationship to carbonic anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared from organic molecules that are considered to be weak binders with low selectivity, yielding conjugates with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine.

Combinatorial Chemical Reengineering of the Alpha Class Glutathione Transferases

Previously, we discovered that human glutathione transferases (hGSTs) from the alpha class can be rapidly and quantitatively modified on a single tyrosine residue (Y9) using thioesters of glutathione (GS-thioesters) as acylating reagents. The current work was aimed at exploring the potential of this site-directed acylation using a combinatorial approach, and for this purpose a panel of 17 GS-thioesters were synthesized in parallel and used in screening experiments with the isoforms hGSTs A1-1, A2-2, A3-3, and A4-4. Through analytical HPLC and MALDI-MS experiments, we found that between 70 and 80% of the reagents are accepted and this is thus a very versatile reaction. The range of ligands that can be used to covalently reprogram these proteins is now expanded to include functionalities such as fluorescent groups, a photochemical probe, and an aldehyde as a handle for further chemical derivatization. This site-specific modification reaction thus allows us to create novel functional proteins with a great variety of artificial chemical groups in order to, for example, specifically tag GSTs in biological samples or create novel enzymatic function using appropriate GS-thioesters.

Hydrated and dehydrated tertiary interactions--opening and closing--of a four-helix bundle peptide.

The structural heterogeneity and thermal denaturation of a dansyl-labeled four-helix bundle homodimeric peptide was studied with steady-state and time-resolved fluorescence spectroscopy and with circular dichroism (CD). At room temperature the fluorescence decay of the polarity-sensitive dansyl, located in the hydrophobic core region, can be described by a broad distribution of fluorescence lifetimes, reflecting the heterogeneous microenvironment. However, the lifetime distribution is nearly bimodal, which we ascribe to the presence of two major conformational subgroups. Since the fluorescence lifetime reflects the water content of the four-helix bundle conformations, we can use the lifetime analysis to monitor the change in hydration state of the hydrophobic core of the four-helix bundle. Increasing the temperature from 9 degrees C to 23 degrees C leads to an increased population of molten-globule-like conformations with a less ordered helical backbone structure. The fluorescence emission maximum remains constant in this temperature interval, and the hydrophobic core is not strongly affected. Above 30 degrees C the structural dynamics involve transient openings of the four-helix bundle structure, as evidenced by the emergence of a water-quenched component and less negative CD. Above 60 degrees C the homodimer starts to dissociate, as shown by the increasing loss of CD and narrow, short-lived fluorescence lifetime distributions.

A promiscuous glutathione transferase transformed into a selective thiolester hydrolase.

Human glutathione transferase A1-1 (hGST A1-1) can be reengineered by rational design into a catalyst for thiolester hydrolysis with a catalytic proficiency of 1.4 x 10(7) M(-1). The thiolester hydrolase, A216H that was obtained by the introduction of a single histidine residue at position 216 catalyzed the hydrolysis of a substrate termed GSB, a thiolester of glutathione and benzoic acid. Here we investigate the substrate requirements of this designed enzyme by screening a thiolester library. We found that only two thiolesters out of 18 were substrates for A216H. The A216H-catalyzed hydrolysis of GS-2 (thiolester of glutathione and naphthalenecarboxylic acid) exhibits a k(cat) of 0.0032 min(-1) and a KM of 41 microM. The previously reported catalysis of GSB has a k(cat) of 0.00078 min(-1) and KM of 5 microM. The k(cat) for A216H-catalyzed hydrolysis of GS-2 is thus 4.1 times higher than for GSB. The catalytic proficiency (k(cat)/KM)/k(uncat) for GS-2 is 3 x 10(6) M(-1). The promiscuous feature of the wt protein towards a range of different substrates has not been conserved in A216H but we have obtained a selective enzyme with high demands on the substrate.