5/2/2023 0 Comments Primordia clarityThe fraction of folded protein was calculated from the far-UV CD spectra at 208 nm, taken between 0 and 80☌ a similar profile was obtained at 222 nm (data not shown). (C) T m curves show that Msx1HD(R31P) has a lower T m (33☌) than Msx1HD (53☌) or Msx1HD(R31A) (58☌). (B) Near-UV CD spec- tra (250 to 320 nm) show that Msx1HD(R31P) has reduced absorbance in the characteristic tyrosine and phenylalanine regions (260 and 280 nm, respectively), whereas the tryptophan peak (290 nm) is similar for all three proteins. This is consistent with the known helix-disrupting propensity of pro- line and the helix-promoting propensity of alanine (22). (A) Far-UV CD spectra (200 to 250 nm) show that the ␣ -helical content of Msx1HD(R31P) (56%) is less than that of Msx1HD (65%), whereas Msx1HD(R31A) (71%) has greater ␣ -heli- cal content. CD spectra were col- lected by using the purified Msx1 homeodomain polypeptides (Fig. Molecular mass standards (shown in panel A by the marker lane and in panel B by dashes) are phosphorylase B (100 kDa), bovine serum albumin (77 kDa), ovalbumin (48.2 kDa), carbonic anhydrase (33.8 kDa), soybean trypsin inhibitor (28.6 kDa), and lysozyme (20.5 kDa).ĬD analysis demonstrates altered structure and reduced stability of Msx1HD(R31P) relative to Msx1HD and Msx1HD(R31A). Note that the R31P substitution results in a more slowly migrating protein. The lysates were separated on a 10% polyacrylamide gel, and the Msx1 proteins, which were Myc tagged, were detected with a monoclonal antibody against the Myc epitope. Cell lysates were prepared from CEFs that were not infected (NA) or that were infected with a retrovirus expressing Msx1, Msx1(R31P), or Msx1(R31A). (B) Western blot assay demonstrates the equivalent expression of the indicated Msx1 proteins in infected CEFs. Each protein (2.5 g) was separated on a 15% polyacrylamide gel and visualized by staining with Coomassie brilliant blue. (A) Sodium dodecyl sulfate-polyacryl- amide gel electrophoresis demonstrates the purity of the recombinant Msx1 homeodomain polypeptides Msx1HD, Msx1HD(R31P), and Msx1HD(R31A). Because Msx1(R31P) appears to be inactive and does not affect the action of wild-type Msx1, we propose that the phenotype of affected individuals with selective tooth agenesis is due to haploinsufficiency.Įxpression of Msx1 proteins. Furthermore, Msx1(R31P) does not antagonize the activity of wild-type Msx1 in any of these assays. In assays of ectopic expression in the limb. Is inactive in vivo, since it does not display the activities of wild-type As a consequence, the biochemical activities of Msx1(R31P) are severely impaired, since it exhibits little or no ability to interact with DNA or other protein factors or to function in transcriptional repression. We show that Msx1(R31P) has perturbed structure and reduced thermostability compared with wild-type Msx1. To determine whether the tooth agenesis phenotype may result from haploinsufficiency or a dominant-negative mechanism, we have performed biochemical and functional analyses of the mutant protein Msx1(R31P). Previously, we found that the cause of autosomal dominant selective tooth agenesis in one family is a missense mutation resulting in an arginine-to-proline substitution in the homeodomain of MSX1.
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