The folding and unfolding reactions of the SH3 domain of spectrin can be described by a two-state model. This domain is a beta-sheet barrel containing 62 amino acids. Equilibrium unfolding by urea, guanidine hydrochloride, and heat is completely reversible at pH values below 4.0. At higher pH values the unfolding is reversible as long as the protein concentration is below 1 mg/mL. The Gibbs energy of unfolding in the absence of denaturant, delta GH2O, at pH 3.5 and 298 K is calculated to be 12 kJ mol-1 for urea, chemical, and temperature denaturation. The stability of the protein does not change noticeably between pH 5.0 and 7.0 and is around 15.5 kJ mol-1. Since heat effects of unfolding are relatively small and, as a result, heat-induced melting occurs in a wide temperature range, the analysis of scanning calorimetry data was performed taking into account the temperature dependence of unfolding delta Cp. The free energy of unfolding obtained for this domain (delta GH2O = 14 +/- 2 kJ mol-1) was, within experimental error, similar to those obtained in this work by other techniques and with those reported in the literature for small globular proteins. Kinetics of unfolding and refolding at pH 3.5, followed both by fluorescence and by circular dichroism, provide evidence of the simplest folding mechanism consistent with the two-state approximation. A value for delta GH2O = 13 +/- 0.7 kJ mol-1 can be extrapolated from the kinetic data.(ABSTRACT TRUNCATED AT 250 WORDS)

We have analyzed the existence of obligatory steps in the folding reaction of the alpha-spectrin SH3 domain by mutating Asp 48 (D48G), which is at position i+3 of an isolated two-residue type II' beta-turn. Calorimetry and X-ray analysis show an entropic stabilizing effect resulting from local changes at the dihedral angles of the beta-turn. Kinetic analysis of D48G shows that this beta-turn is fully formed in the transition state, while there is no evidence of its formation in an isolated fragment. Introduction of several mutations in the D48G protein reveals that the local stabilization has not significantly altered the transition state ensemble. All these results, together with previous analysis of other alpha-spectrin and src SH3 mutants, indicate that: (i) in the folding reaction there could be obligatory steps which are not necessarily part of the folding nucleus; (ii) transition state ensembles in beta-sheet proteins could be quite defined and conformationally restricted ('mechanic folding nucleus'); and (iii) transition state ensembles in some proteins could be evolutionarily conserved.

Alternative splicing (AS) is one crucial step of gene expression that must be tightly regulated during neurodevelopment. However, the precise timing of developmental splicing switches and the underlying regulatory mechanisms are poorly understood. Here we systematically analyze the temporal regulation of AS in a large number of transcriptome profiles of developing mouse cortices, in vivo purified neuronal subtypes, and neurons differentiated in vitro. Our analysis reveals early-switch and late-switch exons in genes with distinct functions, and these switches accurately define neuronal maturation stages. Integrative modeling suggests that these switches are under direct and combinatorial regulation by distinct sets of neuronal RNA-binding proteins including Nova, Rbfox, Mbnl, and Ptbp. Surprisingly, various neuronal subtypes in the sensory systems lack Nova and/or Rbfox expression. These neurons retain the “immature” splicing program in early-switch exons, affecting numerous synaptic genes. These results provide new insights into the organization and regulation of the neurodevelopmental transcriptome.

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