Chymotrypsin inhibitor, structure

Despite their lack of stabilizing disulfide bridges Potl inhibitors feature a common, stable fold. The N-terminus is coiled, although in some structures a small /3-strand has been identified. After a turn the structure adopts an a-helical structure, followed by a turn and an other /3-strand. The sequence then features an extended turn or loop motif that contains the reactive site of the inhibitor before it proceeds with a /3-strand running almost parallel to the /3-strand after the a-helix. After another turn and coiled motif a short /3-strand antiparallel to the other /3-strands precedes the coiled C-terminus. Usually the N-terminal residue in the reactive site is an acidic residue followed by an aromatic amino acid, that is, tyrosine or phenylalanine. Figure 11 shows the complex of chymotrypsin inhibitor (Cl) 2 with subtilisin, the hexamer of Cl 2 from H. vulgare and a structural comparison with a trypsin inhibitor from Linum usitatissimum ... 

Chymotrypsinogen 480, 481 Chymotrypsin inhibitor 2 (CI2) folding kinetics 544-577, 577 GroEL binding 605 fragments 577, 578, 587, 588, 595 mechanism of folding 576-588 structure 576, 577 Circular dichroism (CD) 193-195 optimal absorbance for signal to noise 212-214... 

Fig. 5. Protein folding. The unfolded polypeptide chain collapses and assembles to form simple structural motifs such as p-sheets and a-helices by nucleation-condensation mechanisms involving the formation of hydrogen bonds and van der Waal s interactions. Small proteins (eg, chymotrypsin inhibitor 2) attain their final (tertiary) structure in this way. Larger proteins and multiple protein assemblies aggregate by recognition and docking of multiple domains (eg, p-barrels, a-helix bundles), often displaying positive cooperativity. Many noncovalent interactions, including hydrogen bonding, van der Waal s and electrostatic interactions, and the hydrophobic effect are exploited to create the final, compact protein assembly. Further structural...

Another cultivation of Microcystis aeruginosa NIVA Cya 43 yielded the new cyanopeptolin 954 (1004), which is a chymotrypsin inhibitor (1041). The newly isolated chlorodysinosin A (1005), from a Dysidea sponge, has been synthesized and its structure confirmed (1042). 

K. Hatanc, M. Kqjima, M. Tanokura, and K, Takahaihi. Solution structure of bromelain inhibitor VI from pineapple stem structural similarity with Bawmnn-Birk hyprin/chymotrypsin inhibitor from soybean. Biochemistry J5 5379 (1996). 

Fig. 19. Stereodrawing of 6-benzyl-3-chloro-2-pyrone bound to the active site of y-chymotrypsin. The structures of the native enzyme (solid lines) and the enzyme-inhibitor complex (clear lines) are overlaid. Reproduced with permission from Ringe et al. (1985).

The seeds of squash plants are rich in a family of trypsin and chymotrypsin inhibitors that are approximately 35 amino acids in size and have been extensively investigated not only for their enzyme inhibitory activity, but also because they are very stable mini-protein scaffolds with applications in protein engineering. The best studied examples are Ecballium elaterium trypsin inhibitor (EETI-II) and Cucurbita maxima trypsin inhibitor (CMTI). Both X-ray and NMR have been used to characterise their structures, which incorporate a cystine-knot motif formed by three conserved disulphide bonds.93 We will describe this motif in more detail in a later section describing the plant cyclotides. 

Figure 2.66 Proposed folding pathway of chymotrypsin inhibitor. Local regions with sufficient structural preference tend to adopt their favored structures initially (1). These structures come together to form a nucleus with a nativelike, but still mobile, structure (4). This structure then fully condenses to form the native, more rigid structure (5). [From A. R. Fersht and V. Daggett. Cell 108 (2002) S73-S82 with permission from Elsevier.]...

W. MSrki, F. Raschdorf, W. Richter, H. Rink, P. Sieber, H. P. Schnebli, and M. Liersch. Isolation and characterization of native and recombinant eglin c from E. coli, selective proteinase inhibitors for human leukocyte elastase, cathepsin G and chymotrypsin. Peptides Structure and Function (C. M. Deber, V. J. Hruby, and K. D. Kopple, eds.). Pierce Chemical, Rockford, Illinois, 1985, p. 385. 

Next, in Section 8.3.2, we describe long-time full atomic MD simulations (longer than 150 ns) for a small peptide, met-enkephalin (M-Enk), in addition to a larger chymotrypsin inhibitor 2 (CI2), both in ectoine aqueous solutions with the same concentration and in pure water at room temperature. To determine the spatial distribution of each solvent component, the atom number densities of water molecules and ectoine molecules around both solutes were analyzed. We found that one dominant structure of M-Enk does not preferentially exclude the ectoine molecules from its surface as CI2 does. To understand the reason for this difference in ectoine exclusion, in addition to the effect of direct interaction between M-Enk and ectoine, the influence of hydration (i.e., property alteration of the hydration layer near the solute surface) on the development of ectoine preferential exclusion around each solute was examined at the molecular level. 

Hydration Structure around Chymotrypsin Inhibitor 2 and Met-Enkephalin... 

Third, to obtain the spatial profiles for the preferential solvation around a solute molecule, we proposed another definition of the KB integral around the solute molecule with finite dimensions, i.e., the surficial KB integral, to systematically examine the solvation on the distance from the molecular surface of the solute molecule. With the aid of surficial KB treatment, the present MD simulations of chymotrypsin inhibitor 2 (CI2) showed the preferential exclusion of ectoine (i.e., a CS) near the protein surface, while it is generally considered that CSs are preferentially excluded from the surface of proteins [28,29], On the other hand, the preferential exclusion of ectoine was found to be significantly weakened near the surface of much smaller solute met-enkephalin (M-Enk) in its bent structure [39,40]. 

Deniz etal. [82 ] applied spFRET in one of the first studies that demonstrated its use as a potential structural probe revealing heterogeneity of proteins in solution. Chymotrypsin inhibitor 2 (CI2) was FRET labelled with the dye pair TMR (tetramethylrhodamine) and Cy5 (see Chapter 4). Single molecule diffusion experiments were then performed as a function of chemical denaturant. The folded and unfolded subpopulations of the protein at various denaturant concentrations were resolved and the populations of each state (estimated by the relative area of each peak) were shown to be in broad agreement with the supporting ensemble measurements. Further, changes were seen in the mean FRET efficiency of the unfolded distribution with increasing denaturant. 

L. S. Itzhaki, D. E. Otzen, and A. R. Fersht, 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. J. Mol. Biol. 254, 260-288 (1995). 

The structure of chymotrypsin inhibitor 2 (CI2) is particularly well-characterized, but the crystal structure and NMR solution structure have small but distinct differences. Early NMR results suggested that there is an additional pair of anti-parallel -strands in comparison with the crystal structure. Subsequent experiment and constrained simulation concluded that the difference between these experimental techniques is caused by the refinement methodology used. 

A. Li and V. Daggett, Prorei g., 8,1117 (1995). Investigation of the Solution Structure of Chymotrypsin Inhibitor 2 using Molecular Dynamics Comparison to X-ray Crystallographic and NMR Data. 

Fujinaga, M., et al. Crystal and molecular structures of the complex of a-chymotrypsin with its inhibitor turkey ovomucoid third domain at 1.8 A resolution. 

Irreversible inhibition is probably due to the alkylation of a histidine residue.43 Chymotrypsin is selectively inactivated with no or poor inhibition of human leukocyte elastase (HLE) with a major difference the inactivation of HLE is transient.42,43 The calculated intrinsic reactivity of the coumarin derivatives, using a model of a nucleophilic reaction between the ligand and the methanol-water pair, indicates that the inhibitor potency cannot be explained solely by differences in the reactivity of the lactonic carbonyl group toward the nucleophilic attack 43 Studies on pyridyl esters of 6-(chloromethyl)-2-oxo-2//-1 -benzopyran-3-carboxylic acid (5 and 6, Fig. 11.5) and related structures having various substituents at the 6-position (7, Fig. 11.5) revealed that compounds 5 and 6 are powerful inhibitors of human leukocyte elastase and a-chymotrypsin thrombin is inhibited in some cases whereas trypsin is not inhibited.21... 

A few other helical conformations occur occasionally in globular protein structures. The polyproline helix, of the same sort as one strand out of a collagen structure, has been found in pancreatic trypsin inhibitor (Huber et al., 1971) and in cytochrome c551 (Almassy and Dickerson, 1978). An extended e helix has been described as occurring in chymotrypsin (Srinivasan et al., 1976). In view of the usual variability and irregularity seen in local protein conformation it is unclear that either of these last two helix types is reliably distinguishable from simply an isolated extended strand however, the presence of prolines can justify the designation of polyproline helix. 

Steitz TA, Henderson R, Blow DM. 1969. Structure of crystalline a-chymotrypsin. 3. Crystallographic studies of substartes and inhibitors bound to the active site of a-chymotrypsin. J Mol Biol 46 337-348. 

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