Biology subjects

Hamborg, L.

Publications and source records attributed to Hamborg, L..

4 recordsLinked to original sources

bioRxiv2020

Synergistic stabilization of a double mutant in CI2 from an in-cell library screen

Most single point mutations destabilize folded proteins. Mutations that stabilize a protein typically only have a small effect and multiple mutations are often needed to substantially increase the stability. Multiple point mutations may act synergistically on the stability, and it is not straightforward to predict their combined effect from the individual contributions. Here, we have applied an efficient in-cell assay to select variants of the barley chymotrypsin inhibitor 2 with increased stability. We find two variants that are more than 3.8 kJ/mol more stable than the wild-type. In one case the increased stability is the effect of the single substitution D55G. The other case is a double mutant, L49I/I57V, which is 5.1 kJ/mol more stable than the sum of the effects of the individual mutations. In addition to demonstrating the strength of our selection system for finding stabilizing mutations, our work also demonstrate how subtle conformational effects may modulate stability.

biophysics↗

bioRxiv2020

GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII) is an abundant neuronal signaling protein involved in synaptic plasticity and memory formation1,2. The central hub domain regulates the activity of CaMKII by organizing the holoenzyme complex into functional oligomers3-6. Recent findings have suggested that the hub is also an allosteric determinant of kinase activity7, and is thus an emerging target for therapies to correct CaMKII dysregulation8,9. However, pharmacological modulation of the hub domain has never been demonstrated. Here we show that stabilization of the CaMKII hub domain confers neuroprotection. By combining photoaffinity labeling and chemical proteomics using small molecule analogs of the natural metabolite {gamma}-hydroxybutyrate (GHB)10 we reveal that CaMKII is the selective target for GHB. We further find that these GHB analogs bind to the hub interior by solving a 2.2 [A] crystal structure of CaMKII with bound ligand. Using differential scanning fluorimetry, we show that binding of ligands to the hub interior increases the thermal stability of hub oligomers in a concentration-dependent manner. Moreover, we demonstrate the functional significance of this hub stabilization by showing substantial neuroprotective effects in cellular excitotoxicity assays and in a mouse model of cerebral ischemia. Together, our results reveal that CaMKII hub stabilization is the mechanism by which GHB provides endogenous neuroprotection and that small-molecule CaMKII-selective ligands have therapeutic potential.

biochemistry↗

bioRxiv2020

A dual-reporter system for investigating and optimizing protein translation and folding in E. coli

Strategies for investigating and optimising the expression and folding of proteins for biotechnological and pharmaceutical purposes are in high demand. Here, we describe a dual-reporter biosensor system that simultaneously assesses in vivo protein translation and protein folding, thereby enabling rapid screening of mutant libraries. We have validated the dual-reporter system on five different proteins and find an excellent correlation between reporter intensity signals and the levels of protein expression and solubility of the proteins. We further demonstrate the applicability of the dual-reporter system as a screening assay for deep mutational scanning experiments. The system enables high throughput selection of protein variants with high expression levels and altered protein stability. Next generation sequencing analysis of the resulting libraries of protein variants show a good correlation between computationally predicted and experimentally determined protein stabilities. We furthermore show that the mutational experimental data obtained using this system may be useful for protein structure calculations.

biochemistry↗

bioRxiv2020

Global analysis of protein stability by temperature and chemical denaturation

The stability of a protein is a fundamental property that determines under which conditions, the protein is functional. Equilibrium unfolding with denaturants requires preparation of several samples and only provides the free energy of folding when performed at a single temperature. The typical sample requirement is around 0.5 - 1 mg of protein. If the stability of many proteins or protein variants needs to be determined, substantial protein production may be needed. Here we have determined the stability of acyl-coenzyme A binding protein at pH 5.3 and chymotrypsin inhibitor 2 at pH 3 and pH 6.25 by combined temperature and denaturant unfolding. We used a setup where tryptophan fluorescence is measured in quartz capillaries where only 10 l is needed. Temperature unfolding of a series of 15 samples at increasing denaturant concentrations provided accurate and precise thermodynamic parameters. We find that the number of samples may be further reduced and less than 10 g of protein in total are needed for reliable stability measurements. For assessment of stability of protein purified in small scale e.g. in micro plate format, our method will be highly applicable. The routine for fitting the experimental data is made available as a python notebook.

biophysics↗