Structure of α-chymotrypsin refined at 1.68 Å resolution

Tsukada, Harumichi; Blow, D. M.

doi:10.1016/0022-2836(85)90314-6

Cited by 288 publications

(193 citation statements)

References 41 publications

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“…According to this equation the radius of the hydrated a-chymotrypsin sphere is between 25 and 26 A. This value is consistent with the dimensions of the monomer of a-chymotrypsin found by X-ray crystallography (Tsukada & Blow, 1985;Cohen et al, 1981). The rise curves of the electric dichroism can also be fitted by single exponentials; however, the quality of least-squares fits is clearly improved by admitting a second exponential according to Al and AI, are the changes of the light intensity at time t and m, respectively.…”

Section: Resultssupporting

confidence: 86%

“…Since a-chymotrypsin has two short segments of a-helix only (Blow, 1971) contribution to the total dipole moment is relatively small. We have considered these moments by the assignment of 3.5 D for each a-helical peptide bond in the direction from 0 to C of the carbonyl groups (Wada, 1976;Hol, 1985). The dominant part of the dipole moment, which results from the ionized residues, has been calculated by two different procedures.…”

Section: Resultsmentioning

confidence: 99%

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The nature of protein dipole moments: experimental and calculated permanent dipole of .alpha.-chymotrypsin

Antosiewicz¹,

Pörschke²

1989

Biochemistry

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The electric dichroism of a-chymotrypsin has been measured in buffers of various pH values and ion compositions. The stationary dichroism obtained as a function of the electric field strength is not compatible with an induced dipole mechanism and clearly shows that a-chymotrypsin is associated with a substantial permanent dipole moment. After correction for the internal directing electric field according to a sphere model, the dipole moment is 1.6 X C m at p H 8.3 (corresponding to 480 D). This value decreases with decreasing pH (to 1.2 X C m at pH 4.2), but is almost independent of the monovalent salt concentration in the range from 2 to 12 m M and of Mg2+ addition up to 1 mM. The assignment of the permanent dipole moment is confirmed by analysis of the dichroism rise curves. The dichroism decay time constants of (31 f 1) ns at 2 OC can be represented by a spherical model with a radius of 25-26 A, which is consistent with the known X-ray structure. The limiting linear dichroism is slightly dependent on the buffer composition and demonstrates subtle variations of the protein structure. As a complement to the experimental results, electric and hydrodynamic parameters of a-chymotrypsin have been calculated according to the known X-ray structure. Bead model simulations provide the center of diffusion, which is used to calculate dipole moments according to the equilibrium charge distribution evaluated from standard pK values. In addition, dipole moments are also calculated according to the Tanford-Kirkwood model, which is modified by a more detailed consideration of solvation energies and evaluated from an equilibrium distribution of protein states generated by Monte Carlo techniques. The static dipole moment obtained by the first procedure is consistent with the experimental results over the whole pH range investigated, whereas the Monte Carlo results agree with the experimental data from pH 8.3 to 5.7. The fluctuating dipole moment evaluated by the second procedure gives rise to a minor contribution only, which is usually less than 20% of the static dipole moment. In summary, experimental and theoretical results are in satisfactory agreement and demonstrate the existence of a substantial "true" permanent dipole moment associated with a-chymotrypsin.%e assessment of electric properties for biological macromolecules proved to be particularly difficult-mainly due to the fact that these molecules usually bear a large number of charged residues. In most cases these residues attract counterions, which form a counterion atmosphere with a particularly high polarizability and thus cause large contributions to the measured electric properties. Although the polarization *Present address:

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Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

The nature of protein dipole moments: experimental and calculated permanent dipole of .alpha.-chymotrypsin

Antosiewicz¹,

Pörschke²

1989

Biochemistry

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“…Three-dimensional models of the structures of cercarial elastase and trophozoite cysteine protease were built following the approach of Blundell and coworkers (16,17). Seven mammalian serine proteases, bovine chymotrypsin (18), porcine pancreatic elastase (19), rat mast cell protease (20), human neutrophil elastase (21), rat tonin (22), porcine kallikrein (23), and bovine trypsin (24), were used to derive a structural alignment for cercarial elastase (25). Papain (26) and actinidin (27) were used for trophozoite cysteine protease.…”

Section: Methodsmentioning

confidence: 99%

Structure-based inhibitor design by using protein models for the development of antiparasitic agents.

Ring

Sun

McKerrow

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

249

125

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The lack of an experimentally determined structure of a target protein frequently limits the application of structure-based drug design methods. In an effort to overcome this limitation, we have investigated the use of computer model-built structures for the identification of previously unknown inhibitors of enzymes from two major protease families, serine and cysteine proteases. We have successfully used our model-built structures to identify computationally and to confirm experimentally the activity of nonpeptidic inhibitors directed against important enzymes in the schistosome [2-(4-methoxybenzoyl)-l-naphthoic acid, Ki = 3 ,uM] and malaria {oxalic bis[(2-hydroxy-1-naphthylmethylene)hydrazide], IC50 = 6 ,uM} parasite life cycles.Proteases are involved in many important biological processes including protein turnover, blood coagulation, complement activation (1), hormone processing (2), and cancer cell invasion (3). Thus, they are frequently chosen as targets for drug design and discovery. Noteworthy examples include the design of angiotensin-converting enzyme inhibitors for the treatment of hypertension (4) and programs to develop human immunodeficiency virus protease inhibitors to block proliferation of the AIDS virus (5). The critical role proteases play in the life cycle of parasitic organisms also makes them attractive drug-design targets for these infectious diseases (6).In the most simple terms, structure-based drug design methods identify favorable and unfavorable interactions between a potential inhibitor and target receptor and maximize the beneficial interactions to increase binding affinity. Obtaining an accurate structure for the receptor or ligandreceptor complex is a logical step in this process. X-ray crystallography continues to be the source of high-resolution information about protein structures. However, considerable delays often exist between determining the sequence of a protein and solving its structure. Difficulties in protein expression and more commonly in protein crystallization can delay x-ray structure determination.Currently, no general method exists for predicting tertiary structure from amino acid sequences. However, when a protein target is homologous to another protein or group of proteins of known structure, a sensible model structure can be proposed. Recent comparisons between model and crystal structures permit an assessment of the overall accuracy expected from homology model-built structures (7-9). For a sequence that is 80% identical to a protein of known structure, the expected rms deviation of the core residues is -0.6 A (10). The expected rms deviation increases to 1.8 A when the sequences are only 20% identical. However, model-built structures could still be useful in finding previously unknown lead compounds despite the uncertainties in the lower part of this range if the errors cluster far away from the enzyme active site.The proteases targeted for inhibitor design in this study are important in establishing schistosome infection or necessary for the mainten...

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“…Eliminating true structural differences in extracting errors There are real differences between multiple molecules in the asymmetric unit due to differences in the packing environment "The four-letter code refers to the PDB designation for each structure, for which the references are: lTHB (Waller & Liddington, 1990); 2CCY (Finzel et al, 1985); 4CHA (Tsukada & Blow, 1985); 4DFR (Bolin et al, 1982); ZHHB (Fermi et al, 1984); 3CYT (Takano & Dickerson, 1980); ZAZA (Baker, 1988); l G D l (Skarzynski et al, 1987); lAZA (Norris et al, 1983);lGPl (Epp et al, 1983); 1HMQ (Stenkamp et al, 1982);2pKA (Bodeetal., 1983); 2PFK (Rypniewski Evans, 1989); 1FCB (Xia & Mathews, 1990); 4MDH (Birktoft et al, 1989); 4ATc (Ke et al, 1984); ~F C I (Deisenhofer, 1981); IHBS (Padlan & Love, 1985). of the independent molecules.…”

Section: Deriving the Empirical Error Curve Ex(b)mentioning

confidence: 99%

Significance of structural changes in proteins: Expected errors in refined protein structures

Stroud

Fauman

1995

Protein Science

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A quantitative expression key to evaluating significant structural differences or induced shifts between any two protein structures is derived. Because crystallography leads to reports of a single (or sometimes dual) position for each atom, the significance of any structural change based on comparison of two structures depends critically on knowing the expected precision of each median atomic position reported, and on extracting it for each atom, from the information provided in the Protein Data Bank and in the publication. The differences between structures of protein molecules that should be identical, and that are normally distributed, indicating that they are not affected by crystal contacts, were analyzed with respect to many potential indicators of structure precision, so as to extract, essentially by "machine learning" principles, a generally applicable expression involving the highest correlates. Eighteen refined crystal structures from the Protein Data Bank, in which there are multiple molecules in the crystallographic asymmetric unit, were selected and compared. The thermal E factor, the connectivity of the atom, and the ratio of the number of reflections to the number of atoms used in refinement correlate best with the magnitude of the positional differences between regions of the structures that otherwise would be expected to be the same. These results are embodied in a six-parameter equation that can be applied to any crystallographically refined structure to estimate the expected uncertainty in position of each atom. Structure change in a macromolecule can thus be referenced to the expected uncertainty in atomic position as reflected in the variance between otherwise identical structures with the observed values of correlated parameters.

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Structure of α-chymotrypsin refined at 1.68 Å resolution

Cited by 288 publications

References 41 publications

The nature of protein dipole moments: experimental and calculated permanent dipole of .alpha.-chymotrypsin

The nature of protein dipole moments: experimental and calculated permanent dipole of .alpha.-chymotrypsin

Structure-based inhibitor design by using protein models for the development of antiparasitic agents.

Significance of structural changes in proteins: Expected errors in refined protein structures

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