12/21/2023 0 Comments Fibrillin 1 calcium ion bonding pubmedThis proposed structural heterogeneity may underlie the observed differences in stability and cellular trafficking of proteins containing such changes. Cysteine substitutions affecting other cbEGF disulfide bonds are likely to have different effects. These data demonstrate that C1977Y and C1977R have localized structural effects, confined to the N-terminal end of the mutant domain, which disrupt domain packing. Fibrillin-1 is a 350-kDa glycoprotein and has a modular structure comprising 47 epidermal growth factor-like (EGF) domains, seven transforming growth factor 1. Further analysis of mutant fragments showed that domain packing of cbEGF29-30, but not cbEGF30-31, was disrupted. Fibrillin-1 is the major constitutive element of extracellular microfibrils encoded by the large FBN1 gene that contains 66 exons spanning 235 kb of genomic DNA on chromosome 15q21.1. Each substitution caused the loss of high affinity calcium binding to cbEGF30, consistent with intradomain misfolding, but the calcium binding properties of cbEGF29 and cbEGF31 were surprisingly unaffected. The domain structure as well as the number and. Analysis of the wild-type fragment identified two high affinity and one low affinity calcium-binding sites. The fibrillin-2 gene (FBN2, previouslyFib5) on chromosome 5q23-q31 is closely related to fibrillin-1 (fig 1). Fibrillin-1 is encoded by FBN1 on human chromosome 15q21 and fibrillin-2 is encoded by FBN2 on 5q23. Limited proteolysis, 1H NMR, and calcium chelation studies have been used to probe the effect of each substitution on cbEGF30 and its flanking domains. Here, we studied pathogenic mutations C1977Y and C1977R, which affect cbEGF30 of human fibrillin-1, in a recombinant three cbEGF domain fragment (cbEGF29-31). The domain bound to calcium with moderate affinity (Kd 0.6 +/- 0.1 mM) with no major changes in structure induced upon calcium binding. The structure of this domain was probed using NMR methods, indicating features characteristic of the known structures of EGF-like domains. Although known to introduce proteolytic susceptibility, the detailed structural consequences of cysteine substitutions in cbEGF domains are unknown. Additionally, copolymerization of Tsk fibrillin 1 with wild-type fibrillin 1 rescues the abnormal morphology of the Tsk/Tsk aggregates. This peptide could be easily oxidized and refolded. The largest group of disease-causing mutations affecting calcium-binding epidermal growth factor-like (cbEGF) domain function in a wide variety of extracellular and transmembrane proteins is that which results in cysteine substitutions.
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