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Your search keyword '"Helfrich, R."' showing total 3 results
Start Over Author "Helfrich, R." Topic cyclosporins
3 results on '"Helfrich, R."'

1. Preliminary characterization of a cloned neutral isoelectric form of the human peptidyl prolyl isomerase cyclophilin.

Author

Holzman TF, Egan DA, Edalji R, Simmer RL, Helfrich R, Taylor A, and Burres NS

Subjects
Amino Acid Isomerases isolation & purification, Amino Acid Isomerases metabolism, Amino Acid Sequence, Animals, Base Sequence, Carrier Proteins isolation & purification, Carrier Proteins metabolism, Cattle, Chromatography, High Pressure Liquid, Cloning, Molecular, Enzyme Activation, Gene Expression, Humans, Isoelectric Focusing, Isoelectric Point, Molecular Sequence Data, Peptidylprolyl Isomerase, Recombinant Proteins metabolism, Amino Acid Isomerases genetics, Carrier Proteins genetics, Cyclosporins metabolism
Abstract

We report the cloning of a neutral isoelectric form of the human peptidyl prolyl isomerase, cyclophilin, its expression in Escherichia coli, and its purification and comparison to bovine thymus cyclophilin. The cloned protein exhibited a pI of approximately 7.8 and formed a simple 1:1 complex with cyclosporin A. This cloned form had a pI similar to that observed for the neutral isoform (pI approximately 7.4) of human splenocyte cyclophilin. The bovine thymus proteins exhibited anomalous behavior on CM-cellulose chromatography but were resolved into alkaline (pI approximately 9.3) isoforms and a new neutral (pI approximately 7.8) isoform by isoelectric focusing gel electrophoresis and ultimately into at least four discrete isoforms by capillary electrophoresis. For cyclosporin A binding we observe a Kd of approximately 160 nM for an electrophoretically heterogeneous preparation of the natural bovine protein and approximately 360 nM for the more homogeneous preparation of the cloned human neutral isoform. Stopped-flow measurements of the activation energies for peptidyl-prolyl isomerase activity indicate the recombinant human protein has an activation enthalpy of 3.67 kcal/mol and an activation entropy of -47.3 cal/K-mol for cis----trans isomerization.

Published
1991

2. Isotope-edited NMR of cyclosporin A bound to cyclophilin: evidence for a trans 9,10 amide bond.

Author

Fesik SW, Gampe RT Jr, Holzman TF, Egan DA, Edalji R, Luly JR, Simmer R, Helfrich R, Kishore V, and Rich DH

Subjects
Amides, Amino Acid Isomerases chemistry, Carbon Isotopes, Carrier Proteins chemistry, Cyclosporins chemistry, Escherichia coli genetics, Humans, Leucine analogs & derivatives, Leucine chemistry, Magnetic Resonance Spectroscopy methods, Peptidylprolyl Isomerase, Phenylalanine chemistry, Protein Binding, Protein Conformation, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Tryptophan chemistry, Amino Acid Isomerases metabolism, Carrier Proteins metabolism, Cyclosporins metabolism
Abstract

The binding of a 13C-labeled cyclosporin A (CsA) analog to cyclophilin (peptidyl prolyl isomerase) was examined by means of isotope-edited nuclear magnetic resonance (NMR) techniques. A trans 9,10 peptide bond was adopted when CsA was bound to cyclophilin, in contrast to the cis 9,10 peptide bond found in the crystalline and solution conformations of CsA. Furthermore, nuclear Overhauser effects (NOEs) were observed between the zeta 3 and epsilon 3 protons of the methylleucine (MeLeu) residue at position 9 of CsA and tryptophan121 (Trp121) and phenylalanine (Phe) protons of cyclophilin, suggesting that the MeLeu9 residue of CsA interacts with cyclophilin. These results illustrate the power of isotope-edited NMR techniques for rapidly providing useful information about the conformations and active site environment of inhibitors bound to their target enzymes.

Published
1990
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3. NMR studies of [U-13C]cyclosporin A bound to cyclophilin: bound conformation and portions of cyclosporin involved in binding

Author

Fesik, S. W., Gampe, R. T., Eaton, H. L., Gemmecker, G., Olejniczak, E. T., Neri, Placido, Holzman, T. F., Egan, D. A., Edalji, R., Simmer, R., Helfrich, R., Hochlowski, J., and Jackson, M.

Subjects
Models, Molecular, Isomerase activity, Magnetic Resonance Spectroscopy, Stereochemistry, Protein Conformation, Molecular Conformation, Cyclosporins, Calorimetry, Biochemistry, Protein structure, Cyclosporin a, Humans, Amino Acid Sequence, Binding site, Cyclophilin, Amino Acid Isomerases, Peptidylprolyl isomerase, Carbon Isotopes, Binding Sites, Chemistry, Carbon-13 NMR, Peptidylprolyl Isomerase, Small molecule, Recombinant Proteins, enzymes and coenzymes (carbohydrates), Carrier Proteins
Abstract

Cyclosporin A (CsA), a potent immunosuppressant, is known to bind with high specificity to cyclophilin (CyP), a 17.7 kDa protein with peptidyl-prolyl isomerase activity. In order to investigate the three-dimensional structure of the CsA/CyP complex, we have applied a variety of multidimensional NMR methods in the study of uniformly 13C-labeled CsA bound to cyclophilin. The 1H and 13C NMR signals of cyclosporin A in the bound state have been assigned, and from a quantitative interpretation of the 3D NOE data, the bound conformation of CsA has been determined. Three-dimensional structures of CsA calculated from the NOE data by using a distance geometry/simulated appealing protocol were found to be very different from previously determined crystalline and solution conformations of uncomplexed CsA. In addition, from CsA/CyP NOEs, the portions of CsA that interact with cyclophilin were identified. For the most part, those CsA residues with NOEs to cyclophilin were the same residues important for cyclophilin binding and immunosuppressive activity as determined from structure/activity relationships. The structural information derived in this study together with the known structure/activity relationships for CsA analogues may prove useful in the design of improved immunosuppressants. Moreover, the approach that is described for obtaining the structural information is widely applicable to the study of small molecule/large molecule interactions.

Published
1991

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