EurekaMag
+ Translate
+ Most Popular
Gaucher's disease;thirty-two years experience at Siriraj Hospital
A study of Macrobathra Meyrick from China (Lepidoptera, Cosmopterigidae)
First occurrence in ores of tetragonal chalcocite
Effects of trace element nutrition on sleep patterns in adult women
N.Z. range management guidelines. 2. Design of grazing management systems for tussock country
A case of lipoma of the esophagus
A revision of world Acanthosomatidae (Heteroptera: Pentatomidae): keys to and descriptions of subfamilies, tribes and genera, with designation of types
Life history of the coronate scyphozoan Linuche unguiculata (Swartz, 1788)
Perceptual restoration of obliterated sounds
Mutagenicity studies on two chromium(III) coordination compounds
The formation of the skeleton. I. Growth of a long bone. 1st appearance of a center of calcification
Leucopenia and abnormal liver function in travellers on malaria chemoprophylaxis
The joint commission: four key root causes loom large in sentinel event data
Treatment of vitiligo with topical 15% lactic acid solution in combination with ultra violet-A
Behaviour of dairy cows within three hours after feed supply: I. Influence of housing type and time elapsing after feed supply
Observations of the propagation velocity and formation mechanism of burst fractures caused by gunshot
Management and control of patients with type 2 diabetes mellitus in Lebanon: results from the International Diabetes Management Practices Study (IDMPS)
The diet composition and nutritional knowledge of patients with anorexia nervosa
Physoporella croatica Herak, 1958 of the Slovak karst Anisian (Slovakia, the West Carpathians Mts.)
Bright lights, big noise. How effective are vehicle warning systems?
Ein Plesiosaurier-Rest mit Magensteinen aus mittlerem Lias von Quedlinburg
Incidence of Chlamydia trachomatis in patients with sterility
Monster soup: the microscope and Victorian fantasy
Preliminary tests with residual sprays against poultry lice
Duration of the life of plants in phylogeny

Recombination of S-peptide with S-protein during folding of ribonuclease S. I. Folding pathways of the slow-folding and fast-folding classes of unfolded S-protein


Recombination of S-peptide with S-protein during folding of ribonuclease S. I. Folding pathways of the slow-folding and fast-folding classes of unfolded S-protein



Journal of Molecular Biology 135(1): 231-244



ISSN/ISBN: 0022-2836

PMID: 43398

DOI: 10.1016/0022-2836(79)90349-8

The refolding kinetics of RNase S were measured by tyrosine absorbance, by tyrosine fluorescence emission and by rapid binding of the specific inhibitor 2'CMP to folded [bovine pancreatic] RNase S. The S-protein is 1st unfolded at pH 1.7 and then either mixed with S-peptide as refolding is initiated by a stopped-flow pH jump to pH 6.8, or the same results are obtained if S-protein and S-peptide are present together before refolding is initiated. The refolding kinetics of RNase S were measured as a function of temperature (10-40.degree. C) and of protein concentration (10-120 .mu.M). The results are compared to the folding kinetics of S-protein alone and to earlier studies of RNase A. A thermal folding transition of S-protein was found below 30.degree. C at pH 1.7. The refolding kinetics of unfolded S-protein as it is found above 30.degree. C at pH 1.7, is characterized together with the kinetics of combination between S-peptide and S-protein during folding at pH 6.8. Two classes of unfolded S-protein molecules are found, fast-folding and slow-folding molecules, in a 20:80 ratio. This is the same result as that found earlier for RNase A; it is expected if the slow-folding molecules are produced by the slow cis-trans isomerization of proline residues after unfolding, since S-protein contains all 4 proline residues of RNase A. The refolding kinetics of the fast-folding molecules show clearly that combination between S-peptide and S-protein occurs before folding of S-protein is complete. If combination occurred only after complete folding, then the kinetics of formation of RNase S should be rather slow (5s and 100s at 30.degree. C) and nearly independent of protein concentration, as shown by separate measurements of the folding kinetics of S-protein, and of the combination between S-peptide and folded S-protein. The observed folding kinetics are faster than predicted by this model and also the folding rate increases strongly with protein concentration (apparent 1.6 order kinetics). The fact that RNase S is formed more rapidly than S-protein alone is sufficient by itself to show that combination with S-peptide precedes complete folding of S-protein. Computer simulation of a simple, parallel-pathway scheme reproduces the folding kinetics of the fast-folding molecules. All 3 probes give the same folding kinetics. These results exclude the model for protein folding in which the rate-limiting step is an initial diffusion of the polypeptide chain into a restricted range of 3-dimensional configurations (nucleation) followed by rapid folding (propagation). If this model were valid, comparable rates of folding for RNase A and for S-protein and no populated folding intermediates, so that combination between S-peptide and S-protein should occur after folding is complete. Instead, RNase A folds 60 times more rapidly than S-protein and also combination with S-peptide occurs before folding of S-protein is complete. The folding rate of S-protein increases after the formation, or stabilization, of an intermediate which results from combination with S-peptide. They support a sequential model for protein folding where the rates of successive steps in folding depend on the stabilites of preceding intermediates. The refolding kinetics of the slow-folding molecules are complex. two results demonstrate the presence of folding intermediates: the 3 probes show different kinetic progress curves, and the folding kinetics are concentration-dependent, in contrast to the results expected if complete folding of S-protein precedes combination with S-peptide. A faster phase of the slow-refolding reaction is detected both by tyrosine absorbance and fluorescence emission but not by 2'CMP binding, indicating that native RNase S is not formed in this phase. Comparison of the kinetic progress curves measured by different probes is made with the use of the kinetic ratio test, which is defined here.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 068518783

Download citation: RISBibTeXText

Related references

Proline isomerization in unfolded ribonuclease A. The equilibrium between fast-folding and slow-folding species is independent of temperature. European Journal of Biochemistry 128(1): 77-80, 1982

Recombination of S-peptide with S-protein during folding of ribonuclease S. II. Kinetic characterization of a stable folding intermediate shown by S-protein at pH 1.7. Journal of Molecular Biology 135(1): 245-254, 1979

Exploring the folding pathways of annexin I, a multidomain protein. II. Hierarchy in domain folding propensities may govern the folding process. Journal of Molecular Biology 279(5): 1177-1185, 1998

Fast-folding and slow-folding forms of unfolded proteins. Methods in Enzymology 131: 70-82, 1986

Is protein folding rate dependent on number of folding stages? Modeling of protein folding with ferredoxin-like fold. Biochemistry. Biokhimiia 75(6): 717-727, 2010

Protein folding as a complex reaction: a two-component potential for the driving force of folding and its variation with folding scenario. Plos one 10(4): E0121640, 2015

Molecular basis of co-operativity in protein folding. III. Structural identification of cooperative folding units and folding intermediates. Journal of Molecular Biology 227(1): 293-306, 1992

The protein folding network indicates that the ultrafast folding mutant of villin headpiece subdomain has a deeper folding funnel. Journal of Chemical Physics 134(20): 205104, 2011

From folding theories to folding proteins: a review and assessment of simulation studies of protein folding and unfolding. Annual Review of Physical Chemistry 52: 499-535, 2001

The influence of intrinsic folding mechanism of an unfolded protein on the coupled folding-binding process during target recognition. Proteins 87(4): 265-275, 2019

Kinetic coupling between protein folding and prolyl isomerization. II. Folding of ribonuclease A and ribonuclease T1. Journal of Molecular Biology 224(1): 231-240, 1992

CD properties of the fast- and slow-folding forms of unfolded ribonuclease A. Febs Letters 139(2): 190-192, 1982

Molecular dissection of the folding mechanism of the a subunit of tryptophan synthase: an amino-terminal autonomous folding unit controls several rate-limiting steps in the folding of a single domain protein. Biochemistry (American Chemical Society) 38(31): 205-14, 1999

Analysis of protein folding by fast protein liquid chromatography. Modular domain folding of gamma-II-crystallin from calf eye-lens. Biological Chemistry Hoppe-Seyler 372(1): 23-26, 1991

Molecular dissection of the folding mechanism of the alpha subunit of tryptophan synthase: an amino-terminal autonomous folding unit controls several rate-limiting steps in the folding of a single domain protein. Biochemistry 38(31): 10205-10214, 1999