Mapping Site-Specific Changes that Affect Stability of the NTerminal Domain of Calmodulin

Biophysical tools have been invaluable in formulating therapeutic proteins. These tools characterize protein stability rapidly in a variety of solution conditions, but in general, the techniques lack the ability to discern site-specific information to probe how solution environment acts to stabilize or destabilize the protein. NMR spectroscopy can provide site-specific information about subtle structural changes of a protein under different conditions, enabling one to assess the mechanism of protein stabilization. In this study, NMR was employed to detect structural perturbations at individual residues as a result of altering pH and ionic strength. The N-terminal domain of calmodulin (N-CaM) was used as a model system, and the 1H-15N heteronuclear single quantum coherence (HSQC) experiment was used to investigate effects of pH and ionic strength on individual residues. NMR analysis revealed that different solution conditions affect individual residues differently, even when the amino acid sequence and structure are highly similar. This study shows that addition of NMR to the formulation toolbox has the ability to extend understanding of the relationship between site-specific changes and overall protein stability

Further clinical and molecular characterization of an XLID syndrome associated with BRWD3 variants, a gene implicated in the leukemia-related JAK-STAT pathway

BACKGROUND: Since the first description of a BRWD3-related phenotype in 2007, 21 additional families have been reported with intellectual disability (ID). METHODS: Using exome sequencing (ES) and international datasharing, we identified 14 new unrelated individuals with a pathogenic BRWD3 variant (12 males and 2 females one with skewed X-inactivation). Including the 31 previously individuals published in the literature with clinical data available, we describe the phenotype of 43 males and 2 females, with 32 different BRWD3 variants. RESULTS: Most common features in males (excluding the one with the mosaic variant) include ID (39/39 males), speech delay (24/25 males), postnatal macrocephaly (28/35 males) with prominent forehead (18/25 males) and large ears (14/26 males), and obesity (12/27 males). Both females present with macrocephaly, speech delay and epilepsy while epilepsy was only observed in 4/41 males. Among the 28 variants with available segregation reported, 19 were inherited from unaffected mothers and 9 were de novo. CONCLUSION: This study demonstrates that the BRWD3-related phenotype could be non-specific, leading to difficulty in clinical recognition of this disorder. A genotype-first approach however allows the diagnosis of the BRWD3-related disorder. The refined clinical features presented here will prove useful for reverse phenotyping