Torleif Härd

Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli

A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subclone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N‐terminal fusion proteins and an N‐terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR‐sized and nonmembrane human proteins as soluble fusion proteins in E. coli.

Stabilization of a beta-hairpin in monomeric Alzheimer's amyloid-beta peptide inhibits amyloid formation.

According to the amyloid hypothesis, the pathogenesis of Alzheimers disease is triggered by the oligomerization and aggregation of the amyloid-β (Aβ) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar Aβ assemblies is accompanied by a conformational change toward a high content of β-structure. Here, we report the solution structure of Aβ(1–40) in complex with the phage-display selected affibody protein ZAβ3, a binding protein of nanomolar affinity. Bound Aβ(1–40) features a β-hairpin comprising residues 17–36, providing the first high-resolution structure of Aβ in β conformation. The positions of the secondary structure elements strongly resemble those observed for fibrillar Aβ. ZAβ3 stabilizes the β-sheet by extending it intermolecularly and by burying both of the mostly nonpolar faces of the Aβ hairpin within a large hydrophobic tunnel-like cavity. Consequently, ZAβ3 acts as a stoichiometric inhibitor of Aβ fibrillation. The selected Aβ conformation allows us to suggest a structural mechanism for amyloid formation based on soluble oligomeric hairpin intermediates.

Stabilization of neurotoxic Alzheimer amyloid-β oligomers by protein engineering

Soluble oligomeric aggregates of the amyloid-β peptide (Aβ) have been implicated in the pathogenesis of Alzheimer’s disease (AD). Although the conformation adopted by Aβ within these aggregates is not known, a β-hairpin conformation is known to be accessible to monomeric Aβ. Here we show that this β-hairpin is a building block of toxic Aβ oligomers by engineering a double-cysteine mutant (called Aβcc) in which the β-hairpin is stabilized by an intramolecular disulfide bond. Aβ40cc and Aβ42cc both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Aβ aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in Aβcc oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that β-sheet-containing Aβ42cc oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Aβ42, in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Aβ oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Aβ42 is more toxic than the shorter Aβ40, and why certain inherited mutations are linked to protofibril formation and early-onset AD.

Autoproteolysis coupled to protein folding in the SEA domain of the membrane-bound MUC1 mucin

The single cell layer of the lungs and the gastrointestinal tract is protected by the mucus formed by large glycoproteins called mucins. Transmembrane mucins typically contain 110-residue SEA domains located next to the membrane. These domains undergo post-translational cleavage between glycine and serine in a characteristic GSVVV sequence, but the two peptides remain tightly associated. We show that the SEA domain of the human MUC1 transmembrane mucin undergoes a novel type of autoproteolysis, which is catalyzed by conformational stress and the conserved serine hydroxyl. We propose that self-cleaving SEA domains have evolved to dissociate as a result of mechanical rather than chemical stress at the apical cell membrane and that this protects epithelial cells from rupture. We further suggest that the cell can register mechanical shear at the mucosal surface if the dissociation is signaled via loss of a SEA-binding protein.

Inhibition of amyloid formation.

Amyloid is aggregated protein in the form of insoluble fibrils. Amyloid deposition in human tissue-amyloidosis-is associated with a number of diseases including all common dementias and type II diabetes. Considerable progress has been made to understand the mechanisms leading to amyloid formation. It is, however, not yet clear by which mechanisms amyloid and protein aggregates formed on the path to amyloid are cytotoxic. Strategies to prevent protein aggregation and amyloid formation are nevertheless, in many cases, promising and even successful. This review covers research on intervention of amyloidosis and highlights several examples of how inhibition of protein aggregation and amyloid formation has been achieved in practice. For instance, rational design can provide drugs that stabilize a native folded state of a protein, protein engineering can provide new binding proteins that sequester monomeric peptides from aggregation, small molecules and peptides can be designed to block aggregation or direct it into non-cytotoxic paths, and monoclonal antibodies have been developed for therapies towards neurodegenerative diseases based on inhibition of amyloid formation and clearance.

Solution structure and DNA-binding properties of a thermostable protein from the archaeon Sulfolobus solfataricus.

The archaeon Sulfolobus solfataricus expresses large amounts of a small basic protein, Sso7d, which was previously identified as a DNA-binding protein possibly involved in compaction of DNA. We have determined the solution structure of Sso7d. The protein consists of a triple-stranded anti-parallel β-sheet onto which an orthogonal double-stranded β-sheet is packed. This topology is very similar to that found in eukaryotic Src homology-3 (SH3) domains. Sso7d binds strongly (Kd < 10 μM) to double-stranded DNA and protects it from thermal denaturation. In addition, we note that ɛ-mono-methylation of lysine side chains of Sso7d is governed by cell growth temperatures, suggesting that methylation is related to the heat-shock response.

His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors

AbstractWe have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative effect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with a C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins. abbreviations: BSA – bovine serum albumin; DBD – DNA binding domain; DCW – dry cell weight; EDTA – ethylenediaminetetraacetic acid; GFP – green fluorescent protein; IMAC – immobilized metal ion affinity chromatography; IPTG – isopropyl-β-d-thiogalactopyranoside; LB – Luria-Bertani; MES – 2-(N-morpholino) ethane sulfonic acid; OD – optical density; ORF – open reading frame; PCR – polymerase chain reaction; SDS-PAGE – sodium dodecyl sulfate polyacrylamide gel electrophoresis.

An affibody in complex with a target protein: Structure and coupled folding

Combinatorial protein engineering provides powerful means for functional selection of novel binding proteins. One class of engineered binding proteins, denoted affibodies, is based on the three-helix scaffold of the Z domain derived from staphylococcal protein A. The ZSPA-1 affibody has been selected from a phage-displayed library as a binder to protein A. ZSPA-1 also binds with micromolar affinity to its own ancestor, the Z domain. We have characterized the ZSPA-1 affibody in its uncomplexed state and determined the solution structure of a Z:ZSPA-1 protein–protein complex. Uncomplexed ZSPA-1 behaves as an aggregation-prone molten globule, but folding occurs on binding, and the original (Z) three-helix bundle scaffold is fully formed in the complex. The structural basis for selection and strong binding is a large interaction interface with tight steric and polar/nonpolar complementarity that directly involves 10 of 13 mutated amino acid residues on ZSPA-1. We also note similarities in how the surface of the Z domain responds by induced fit to binding of ZSPA-1 and Ig Fc, respectively, suggesting that the ZSPA-1 affibody is capable of mimicking the morphology of the natural binding partner for the Z domain.

Structural basis for high-affinity HER2 receptor binding by an engineered protein.

The human epidermal growth factor receptor 2 (HER2) is specifically overexpressed in tumors of several cancers, including an aggressive form of breast cancer. It is therefore a target for both cancer diagnostics and therapy. The 58 amino acid residue Zher2 affibody molecule was previously engineered as a high-affinity binder of HER2. Here we determined the structure of Zher2 in solution and the crystal structure of Zher2 in complex with the HER2 extracellular domain. Zher2 binds to a conformational epitope on HER2 that is distant from those recognized by the therapeutic antibodies trastuzumab and pertuzumab. Its small size and lack of interference may provide Zher2 with advantages for diagnostic use or even for delivery of therapeutic agents to HER2-expressing tumors when trastuzumab or pertuzumab are already employed. Biophysical characterization shows that Zher2 is thermodynamically stable in the folded state yet undergoing conformational interconversion on a submillisecond time scale. The data suggest that it is the HER2-binding conformation that is formed transiently prior to binding. Still, binding is very strong with a dissociation constant KD = 22 pM, and perfect conformational homogeneity is therefore not necessarily required in engineered binding proteins. A comparison of the original Z domain scaffold to free and bound Zher2 structures reveals how high-affinity binding has evolved during selection and affinity maturation and suggests how a compromise between binding surface optimization and stability and dynamics of the unbound state has been reached.

Ansig for Windows: An interactive computer program for semiautomatic assignment of protein NMR spectra

Assignment of NMR spectra is a prerequisite for structure determination of proteins using NMR. The time spent on the assignment is comparatively long compared to that spent on other parts in the protein structure determination process, but it can be shortened by using either interactive or fully automated computer programs. To benefit from the advantages of both types of program we have developed a version of the interactive assignment program ANSIG to include automatized, yet user-supervised, routines. The new program includes tools for (i) semiautomatic sequential assignment, (ii) plotting of distances from PDB structure files directly in NMR spectra and (iii) statistical analysis of distance restraint violations with the possibility to directly zoom to violated NOEs in NOESY spectra.