Slow-Folding Steps

RNase T1 as Model System ro Probe Catalysis of Folding 

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As the fold of the RNA is critical, the folding step after purification of the RNA is important. The most commonly used folding protocols involve heat-cooling the RNA in a metal-free buffer such as Tris-EDTA and subsequently adding metals (Ke and Doudna, 2004) such as monovalent or divalent cations, most commonly Na/K+ and Mg2+. Slow renaturation of the folded RNA from denaturing conditions may also be effective, particularly in RNA-protein systems. Both the human 5 virus ribozyme... 

The refolding of the U molecules involves slow steps that are limited by prolyl peptide bond isomerization. Folding steps and prolyl isomerization steps can be mutually interdependent. On the one hand the presence of incorrect isomers in the chain can decelerate crucial folding steps, and on the other hand rapid chain folding can affect... 

Most of the small proteins that were used initially as substrates to test the function of prolyl isomerases contained disulfide bonds, which were left intact during unfolding and refolding. These proteins were used because their unfolding is reversible under a wide variety of conditions and because good evidence existed for a number of them that prolyl isomerizations were involved as rate-limiting steps in their slow-folding reactions. A protein chain without disulfides should be a better model... 

Li, H. L. and Frieden, C. (2007) Observation of sequential steps in the folding of intestinal fatty acid binding protein using a slow folding mutant and F-19 NMR. Proceedings of the National Academy of Sciences of the U. S. A., 104(29), 11993-11998. 

In the native protein these less stable ds-proline peptides are stabilized by the tertiary structure but in the unfolded state these constraints are relaxed and there is an equilibrium between ds- and trans-isomers at each peptide bond. When the protein is refolded a substantial fraction of the molecules have one or more proline-peptide bonds in the incorrect form and the greater the number of proline residues the greater the fraction of such molecules. Cis-trans isomerization of proline peptides is intrinsically a slow process and in vitro it is frequently the rate-limiting step in folding for those molecules that have been trapped in a folding intermediate with the wrong isomer. 

The steps leading to the formation of the intrinsic chro-mophore have recently been investigated kinetically with S65T-GFP. The process of chromophore formation is an ordered sequence of three distinct steps (1) slow protein folding (kf = 2.44 X 10 s ) that precedes chromophore modification (2) an intermediate step occurs that includes, but may not be necessarily limited to, cycli-zation of the tripeptide chromophore motif (kc = 3.8 X 10 s ) and (3) rate-limiting oxidation of the cyclized chromophore (kox = 1 51 X s ). Reid and Flynn also reasoned that because chromophore forms de novo from purified denatured protein and is a first-order process, GFP chromophore formation is likely to be an autocatalytic process. 

Another kind of slowness comes from the approximately 1000-fold disparity between bonded and nonbonded forces among atoms. This means that a typical covalent bond undergoes about 30 smal1-amplitude, nearly-harmon-ic vibrations in the time required for any other significant molecular motion to take place. In doing dynamics calculations, these fast vibrational modes are a nuisance because they force the use of a very short time step, about. 001 psec. or less. Fortunately, they... 

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