The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: evidence for a nucleation-condensation mechanism for protein folding.


Abstract

The 64-residue protein chymotrypsin inhibitor 2 (CI2) is a single module of structure. It folds and unfolds as a single co-operative unit by simple two-state kinetics via a single rate determining transition state. This transition state has been characterized at the level of individual residues by analysis of the rates and equilibria of folding of some 100 mutants strategically distributed at 45 sites throughout the protein. Only one residue, a helical residue (Ala16) buried in the hydrophobic core, has its full native interaction energy in the transition state. The only region of structure which is well developed in the transition state is the alpha-helix (residues 12 to 24). But, the interactions within it are weakened, especially at the C-terminal region. The rest of the protein has varying degrees of weakly formed structure. Thus, secondary and tertiary interactions appear to form concurrently. These data, reinforced by studies on the structures of peptide fragments, fit a "nucleation-condensation" model in which the overall structure condenses around an element of structure, the nucleus, that itself consolidates during the condensation. The high energy transition state is composed of the whole of the molecule making a variety of weak interactions, the nucleus being those residues that make the strongest interactions. The nucleus here is part of the alpha-helix and some distant residues in the sequence with which it makes contacts. The remainder of the protein has to be sufficiently ordered that it provides the necessary interactions to stabilize the nucleus. The nucleus is only weakly formed in the denatured state but develops in the transition state. The onrush of stability as the nucleus consolidates its local and long range interactions is so rapid that it is not yet fully formed in the transition state. The formation of the nucleus is thus coupled with the condensation. These results are consistent with a recent simulation of the folding of a computer model protein on a lattice which is found to proceed by a nucleation-growth mechanism. We suggest that the mechanism of folding of CI2 may be a common theme in protein folding whereby fundamental folding units of larger proteins, which are modelled by the folding of CI2, form by nucleation-condensation events and coalesce, perhaps in a hierarchical manner. Study holds ProTherm entries: 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958 Extra Details: dG and ddG were measured in the presence of [GdnHCl]50% protein folding; transition state; CI2; barnase;,protein engineering

Submission Details

ID: uML467Mo3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:18 p.m.

Version: 1

Publication Details
Itzhaki LS;Otzen DE;Fersht AR,J. Mol. Biol. (1995) The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: evidence for a nucleation-condensation mechanism for protein folding. PMID:7490748
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3CI2 1993-10-31 REFINEMENT OF THE THREE-DIMENSIONAL SOLUTION STRUCTURE OF BARLEY SERINE PROTEINASE INHIBITOR 2 AND COMPARISON WITH THE STRUCTURES IN CRYSTALS
1CIR 1996-01-29 COMPLEX OF TWO FRAGMENTS OF CI2 [(1-40)(DOT)(41-64)]
1CIS 1993-10-31 CONTEXT DEPENDENCE OF PROTEIN SECONDARY STRUCTURE FORMATION. THE THREE-DIMENSIONAL STRUCTURE AND STABILITY OF A HYBRID BETWEEN CHYMOTRYPSIN INHIBITOR 2 AND HELIX E FROM SUBTILISIN CARLSBERG
1LW6 2002-08-21 1.5 Crystal Structure of the Complex of Subtilisin BPN' with Chymotrypsin Inhibitor 2 at 1.5 Angstrom Resolution
1YPC 1994-01-31 1.7 DIRECT OBSERVATION OF BETTER HYDRATION AT THE N-TERMINUS OF AN ALPHA-HELIX WITH GLYCINE RATHER THAN ALANINE AS N-CAP
5FFN 2016-05-18 1.8 Complex of subtilase SubTY from Bacillus sp. TY145 with chymotrypsin inhibitor CI2A
1CQ4 1998-11-25 1.8 CI2 MUTANT WITH TETRAGLUTAMINE (MGQQQQGM) REPLACING MET59
5FBZ 2016-05-18 1.9 Structure of subtilase SubHal from Bacillus halmapalus - complex with chymotrypsin inhibitor CI2A
2CI2 1988-09-07 2.0 CRYSTAL AND MOLECULAR STRUCTURE OF THE SERINE PROTEINASE INHIBITOR CI-2 FROM BARLEY SEEDS
1YPB 1994-01-31 2.0 DIRECT OBSERVATION OF BETTER HYDRATION AT THE N-TERMINUS OF AN ALPHA-HELIX WITH GLYCINE RATHER THAN ALANINE AS N-CAP
1YPA 1994-01-31 2.0 DIRECT OBSERVATION OF BETTER HYDRATION AT THE N-TERMINUS OF AN ALPHA-HELIX WITH GLYCINE RATHER THAN ALANINE AS N-CAP
2SNI 1988-09-07 2.1 STRUCTURAL COMPARISON OF TWO SERINE PROTEINASE-PROTEIN INHIBITOR COMPLEXES. EGLIN-C-SUBTILISIN CARLSBERG AND CI-2-SUBTILISIN NOVO
1COA 1994-01-31 2.2 THE EFFECT OF CAVITY CREATING MUTATIONS IN THE HYDROPHOBIC CORE OF CHYMOTRYPSIN INHIBITOR 2
1CIQ 1996-03-08 2.2 COMPLEX OF TWO FRAGMENTS OF CI2, RESIDUES 1-40 AND 41-64

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
93.7 Subtilisin-chymotrypsin inhibitor-2A P08626 ICI3_HORVU
100.0 Subtilisin-chymotrypsin inhibitor-2A P01053 ICI2_HORVU
90.5 Subtilisin-chymotrypsin inhibitor-2A P82977 ICIW_WHEAT