Biomaterial Database

Influence of primary sequence transpositions on the folding pathways of ribonuclease T1. 1996

J L Johnson, and F M Raushel

Department of Chemistry, Texas A & M University, College Station 77843, USA.

The slow folding of circularly permuted variants of ribonuclease T1 has been examined using steady-state and frequency-domain fluorescence spectroscopy. The sequence transpositions have previously been designed by eliminating a restrictive Cys2-Cys10 disulfide bond, adjoining the original termini with a three-peptide Gly-Gly-Gly linker, and conferring new termini to four different solvent-exposed beta-turns interposing secondary structural elements [Garrett, J. B., Mullins, L. S., & Raushel, F. M. (1996) Protein Sci. 5, 204-211]. Each of the mutant proteins continues to be rate-limited in folding by the slow trans to cis isomerizations of Pro39 and Pro55, giving rise to a branched mechanism populated by intermediates with mixed proline isomers. However, the overall rate of folding is increased in accordance with the general destabilizing effect of each circular permutation. Steric hindrances imposed by Trp59 on the isomerization around the Tyr38-Pro39 peptide bond have been implicated in decelerating the folding of RNase T1 [Kiefhaber, T., Grunert, H.-P., Hahn, U., & Schmid, F. X. (1992) Proteins: Struct., Funct., Genet. 12, 171-179]; it is this tertiary restraint which appears to be variably relieved by the sequence transpositions. A fluorescence characterization of Trp59 indicates little difference between fully folded RNase T1 and the variants in terms of its lifetime, accessibility to quenchers, and rotational properties. Yet, within protein that is "completely" denatured, Trp59 exhibits variable flexibility, greatest within the circularly permuted variants folding the fastest. Such differences in the dynamic properties of Trp59 between each denatured protein may be direct evidence for a relative loosening of the tertiary fold maintaining the "deleterious" Trp59-Pro39 interaction in the partially folded intermediates.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011489	Protein Denaturation	Disruption of the non-covalent bonds and/or disulfide bonds responsible for maintaining the three-dimensional shape and activity of the native protein.	Denaturation, Protein,Denaturations, Protein,Protein Denaturations
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002151	Calorimetry	The measurement of the quantity of heat involved in various processes, such as chemical reactions, changes of state, and formations of solutions, or in the determination of the heat capacities of substances. The fundamental unit of measurement is the joule or the calorie (4.184 joules). (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D003545	Cysteine	A thiol-containing non-essential amino acid that is oxidized to form CYSTINE.	Cysteine Hydrochloride,Half-Cystine,L-Cysteine,Zinc Cysteinate,Half Cystine,L Cysteine
D004220	Disulfides	Chemical groups containing the covalent disulfide bonds -S-S-. The sulfur atoms can be bound to inorganic or organic moieties.	Disulfide
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005998	Glycine	A non-essential amino acid. It is found primarily in gelatin and silk fibroin and used therapeutically as a nutrient. It is also a fast inhibitory neurotransmitter.	Aminoacetic Acid,Glycine, Monopotassium Salt,Glycine Carbonate (1:1), Monosodium Salt,Glycine Carbonate (2:1), Monolithium Salt,Glycine Carbonate (2:1), Monopotassium Salt,Glycine Carbonate (2:1), Monosodium Salt,Glycine Hydrochloride,Glycine Hydrochloride (2:1),Glycine Phosphate,Glycine Phosphate (1:1),Glycine Sulfate (3:1),Glycine, Calcium Salt,Glycine, Calcium Salt (2:1),Glycine, Cobalt Salt,Glycine, Copper Salt,Glycine, Monoammonium Salt,Glycine, Monosodium Salt,Glycine, Sodium Hydrogen Carbonate,Acid, Aminoacetic,Calcium Salt Glycine,Cobalt Salt Glycine,Copper Salt Glycine,Hydrochloride, Glycine,Monoammonium Salt Glycine,Monopotassium Salt Glycine,Monosodium Salt Glycine,Phosphate, Glycine,Salt Glycine, Monoammonium,Salt Glycine, Monopotassium,Salt Glycine, Monosodium