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Protein science 30(1), 201 - 217 (2021). doi:10.1002/pro.3986, Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited by sample consumption or compatibility with sample buffer components. Here we describe how miniaturized measurement of intrinsic tryptophan fluorescence (NanoDSF assay) in combination with a simplified description of protein unfolding can be used to interrogate the stability of a protein sample. We demonstrate that improved protein stability measures, such as apparent Gibbs free energy of unfolding, rather than melting temperature T$_m$, should be used to rank the results of thermostability screens. The assay is compatible with protein samples of any composition, including protein complexes and membrane proteins. Our data analysis software, MoltenProt, provides an easy and robust way to perform characterization of multiple samples. Potential applications of MoltenProt and NanoDSF include buffer and construct optimization for X‐ray crystallography and cryo‐electron microscopy, screening for small‐molecule binding partners and comparison of effects of point mutations., Published by Protein Society, Bethesda, Md.

Objective: Pantothenate kinase 2-associated neurodegeneration (PKAN) is a rare neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene. PKAN is associated with iron deposition in the basal ganglia and, occasionally, with the occurrence of misshaped erythrocytes (acanthocytes). The aim of this study was to assess residual activity of PANK2 in erythrocytes of PKAN patients and to correlate these data with the type of PANK2 mutations and the progression of neurodegeneration. Methods: Residual PANK2 activities in erythrocytes of 14 PKAN patients and 14 related carriers were assessed by a radiometric assay. Clinical data on neurodegeneration included the Barry-Albright Dystonia Scale (BAD-Scale) besides further general patient features. A molecular visualization and analysis program was used to rationalize the influence of the PKAN causing mutations on a molecular level. Results: Erythrocytes of PKAN patients had markedly reduced or no PANK2 activity. However, patients with at least one allele of the c.1583C > T (T528M) or the c.833G > T (R278L) variant exhibited 12-56% of residual PANK2 activity. In line, molecular modeling indicated only minor effects on enzyme structure for these point mutations. On average, these patients with c.1583C > T or c.833G > T variant had lower BAD scores corresponding to lower symptom severity than patients with other PANK2 point mutations. Interpretation: Residual erythrocyte PANK2 activity could be a predictor for the progression of neurodegeneration in PKAN patients. Erythrocytes are an interesting patient-derived cell system with still underestimated diagnostic potential.

Mutations in the actin cross-linking protein actinin-1 were recently linked to dominantly inherited congenital macrothrombocytopenia. Here, we report that several disease-associated mutations that are located within the actinin-1 actin-binding domain cause increased binding of actinin-1 to actin filaments and enhance filament bundling in vitro. These actinin-1 mutants are also more stably associated with the cytoskeleton in cultured cells, as assessed by biochemical fractionation and fluorescence recovery after photobleaching experiments. For two mutations the disruption of contacts between the calponin homology domains within the actinin actin-binding domain may explain increased filament binding--providing mechanistic and structural insights into the basis of actinin-1 dysfunction in congenital macrothrombocytopenia.