There is a great deal of interest in developing small stably folded miniature proteins. A limited number of these molecules have been described, however they typically have not been characterized in depth. In particular, almost no detailed studies of the thermodynamics and folding kinetics of these proteins have been reported. Here we describe detailed studies of the thermodynamics and kinetics of folding of a 39 residue mixed alpha-beta protein (NTL9(1-39)) derived from the N-terminal domain of the ribosomal protein L9. The protein folds cooperatively and rapidly in a two-state fashion to a native state typical of those found for normal globular proteins. At pH 5.4 in 20mM sodium acetate, 100mM NaCl the temperature of maximum stability is 6 degrees C, the t(m) is 65.3 degrees C, deltaH degrees (t(m)) is between 24.6 kcalmol(-1) and 26.3 kcalmol(-1), and deltaC(p) degrees is 0.38 kcalmol(-1)deg(-1). The thermodynamic parameters are in the range expected on the basis of per residue values determined from databases of globular proteins. H/2H exchange measurements reveal a set of amides that exchange via global unfolding, exactly as expected for a normal cooperatively folded globular protein. Kinetic measurements show that folding is two-state folding. The folding rate is 640 s(-1) and the value of deltaG degrees calculated from the folding and unfolding rates is in excellent agreement with the equilibrium value. A designed thermostable variant, generated by mutating K12 to M, was characterized and found to have a t(m) of 82 degrees C. Equilibrium and kinetic measurements demonstrate that its folding is cooperative and two-state. Study holds ProTherm entries: 15983, 15984, 15985, 15986, 15987, 15988, 15989 Extra Details: A peptide with an amidated C terminus corresponding to the first 39 residues of the ribosomal protein L9 protein folding; L9; protein thermodynamics; protein stability; protein design
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:47 p.m.
|Number of data points||18|
|Proteins||50S ribosomal protein L9 ; 50S ribosomal protein L9|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O buffers:phosphate: 10 mM ; Experimental Assay: dG_H2O buffers:Phosphate: 10 mM ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O buffers:acetate: 20 mM ; Experimental Assay: dCp temp:25.0 C ; Experimental Assay: dG ; Experimental Assay: dCp ; Experimental Assay: Tm ; Experimental Assay: dHvH ; Derived Quantity: ddG_H2O buffers:Phosphate: 10 mM ; Derived Quantity: ddG_H2O buffers:acetate: 20 mM ; Derived Quantity: dTm|
|Libraries||Mutations for sequence MKVIFLKDVKGKGKKGEIKNVADGYANNFLFKQGLAIEATPANLKALEAQKQKEQRQAAEELANAKKLKEQLEKLTVTIPAKAGEGGRLFGSITSKQIAESLQAQHGLKLDKRKIELADAIRALGYTNVPVKLHPEVTATLKVHVTEQK|