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1.
Amino Acids ; 35(2): 511-2, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18704283

ABSTRACT

Recently, Guo et al. have reported structural as well as the binding energy data of the particular interactions between the cleavage sites of hemagglutinin and serine proteases, trypsin and furin, using molecular docking approach. Due to a wrong assignment of protonation state on the histidine, one of the catalytic triad in the active site of both enzymes, their docking results are contradictory with the fundamental principle and previous theoretical studies of the known cleavage mechanism in serine proteases.


Subject(s)
Furin/chemistry , Hemagglutinins, Viral/chemistry , Influenza A Virus, H5N1 Subtype/chemistry , Trypsin/chemistry , Binding Sites , Influenza A Virus, H5N1 Subtype/pathogenicity , Models, Molecular , Molecular Conformation , Serine Endopeptidases/chemistry
2.
Novartis Found Symp ; 245: 146-58; discussion 158-64, 165-8, 2002.
Article in English | MEDLINE | ID: mdl-12027005

ABSTRACT

The fundamental processes that underlie ion channel function are permeation/ selectivity and gating. In an effort to understand ion channel gating, we have used an approach that combines reporter-group spectroscopic techniques (spin labelling/ electron paramagnetic resonance, EPR) and electrophysiological methods with classical biochemical and molecular biological procedures. As an ideal test channel, we have focused our attention on the K+ channel from Streptomyces lividans, KcsA. Through site-directed spin labelling, cysteine chemistry was used to introduce nitroxide radicals into specific sites within KcsA with high reactivity and specificity. EPR spectroscopy analysis of the spin labelled mutants yields two types of structural information: (1) mobility and solvent accessibility of the attached nitroxide through collisional relaxation methods and (2) distances between pairs of nitroxides through dipole-dipole interactions. Using this approach, we analysed the correlation between KcsA crystal structure and the EPR data, extend it to derive low-resolution folds of full-length KcsA and apply it in the determination of the molecular rearrangements responsible for pH-dependent gating.


Subject(s)
Ion Channels/chemistry , Ion Channels/physiology , Bacterial Proteins/chemistry , Bacterial Proteins/physiology , Crystallography, X-Ray , Electron Spin Resonance Spectroscopy/methods , Ion Channel Gating/physiology , Models, Molecular , Potassium Channels/chemistry , Potassium Channels/physiology , Protein Structure, Secondary , Streptomyces/physiology
3.
Biophys J ; 81(5): 2530-46, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11606268

ABSTRACT

We present an approach for calculating conformational changes in membrane proteins using limited distance information. The method, named restraint-driven Cartesian transformations, involves 1) the use of relative distance changes; 2) the systematic sampling of rigid body movements in Cartesian space; 3) a penalty evaluation; and 4) model refinement using energy minimization. As a test case, we have analyzed the structural basis of activation gating in the Streptomyces lividans potassium channel (KcsA). A total of 10 pairs of distance restraints derived from site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR) spectra were used to calculate the open conformation of the second transmembrane domains of KcsA (TM2). The SDSL-EPR based structure reveals a gating mechanism consistent with a scissoring-type motion of the TM2 segments that includes a pivot point near middle of the helix. The present approach considerably reduces the amount of time and effort required to establish the overall nature of conformational changes in membrane proteins. It is expected that this approach can be implemented into restrained molecular dynamics protocol to calculate the structure and conformational changes in a variety of membrane protein systems.


Subject(s)
Bacterial Proteins , Membrane Proteins/chemistry , Models, Molecular , Potassium Channels/chemistry , Electron Spin Resonance Spectroscopy/methods , Molecular Structure , Protein Conformation , Streptomyces/chemistry
4.
Nat Struct Biol ; 8(10): 883-7, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11573095

ABSTRACT

Ion channels catalyze the selective transfer of ions across the membrane in response to a variety of stimuli. These channels gate by controlling the access of ions to a centrally located water-filled pore. The crystal structure of the Streptomyces lividans potassium channel (KcsA) has allowed a molecular exploration of this mechanism. Electron paramagnetic resonance (EPR) studies have uncovered significant conformational changes at the intracellular end of the second transmembrane helix (TM2) upon gating. We have used site-directed spin labeling (SDSL) and EPR spectroscopy in an attempt to quantify the structural rearrangements of the KcsA TM2 bundle underlying the transition from the closed to the open state. Under conditions favoring the closed and open conformations, 10 intersubunit distances were obtained across TM2 segments from tandem dimer constructs. Analysis of these data points to a mechanism in which each TM2 helix tilts away from the permeation pathway, towards the membrane plane, and rotates about its helical axis, supporting a scissoring-type motion with a pivot point near residues 107-108. These movements are accompanied by a large increase in the diameter of the vestibule below the central water-filled cavity.


Subject(s)
Bacterial Proteins , Ion Channel Gating , Potassium Channels/chemistry , Amino Acid Sequence , Dimerization , Electron Spin Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Potassium Channels/physiology , Protein Conformation , Streptomyces/chemistry
5.
Bioorg Med Chem ; 8(5): 1117-28, 2000 May.
Article in English | MEDLINE | ID: mdl-10882022

ABSTRACT

The nature of the interactions between Plasmodium falciparum dihydrofolate reductase (pfDHFR) and antimalarial antifolates, i.e., pyrimethamine (Pyr), cycloguanil (Cyc) and WR99210 including some of their analogues, was investigated by molecular modeling in conjunction with the determination of the inhibition constants (Ki). A three-dimensional structural model of pfDHFR was constructed using multiple sequence alignment and homology modeling procedures, followed by extensive molecular dynamics calculations. Mutations at amino acid residues 16 and 108 known to be associated with antifolate resistance were introduced into the structure, and the interactions of the inhibitors with the enzymes were assessed by docking and molecular dynamics for both wild-type and mutant DHFRs. The Ki values of a number of analogues tested support the validity of the model. A 'steric constraint' hypothesis is proposed to explain the structural basis of the antifolate resistance.


Subject(s)
Drug Resistance , Folic Acid Antagonists/pharmacology , Pyrimethamine/pharmacology , Tetrahydrofolate Dehydrogenase/drug effects , Triazines/pharmacology , Amino Acid Sequence , Folic Acid Antagonists/chemistry , Models, Molecular , Molecular Sequence Data , Molecular Structure , Proguanil , Pyrimethamine/chemistry , Sequence Homology, Amino Acid , Tetrahydrofolate Dehydrogenase/chemistry , Triazines/chemistry
6.
Biochemistry ; 36(29): 8992-9001, 1997 Jul 22.
Article in English | MEDLINE | ID: mdl-9220987

ABSTRACT

The solution structure of oxidized Saccharomycescerevisiae Cys102Ser iso-1-cytochromechas been determined using 1361 meaningful NOEs (of 1676 total) after extending the published proton assignment [Gao, Y., et al. (1990) Biochemistry 29, 6994-7003] to 77% of all proton resonances. The NOE patterns indicate that secondary structure elements are maintained upon oxidation in solution with respect to the solid state and solution structures of the reduced species. Constraints derived from the pseudocontact shifts [diamagnetic reference shift values are those of the reduced protein [Baistrocchi, P., et al. (1996) Biochemistry 35, 13788-13796]] were used in the final stages of structure calculations. After restrained energy minimization with constraints from NOEs and pseudocontact shifts, a family of 20 structures with rmsd values of 0.58 +/- 0.08 and 1.05 +/- 0.10 A (relative to the average structure) for the backbone and all heavy atoms, respectively, was obtained. The solution structure is compared with the crystal structure and the structures of related systems. Twenty-six amide protons were detected in the NMR spectrum 6 days after the oxidized lyophilized protein was dissolved in D2O (pH 7.0 and 303 K); in an analogous experiment, 47 protons were observed in the spectrum of the reduced protein. The decrease in the number of nonexchanging amide protons, which mainly are found in the loop regions 14-26 and 75-82, confirms the greater flexibility of the structure of oxidized cytochrome c in solution. Our finding of increased solvent accessibility in these loop regions is consistent with proposals that an early step in unfolding the oxidized protein is the opening of the 70-85 loop coupled with dissociation of the Met80-iron bond.


Subject(s)
Cytochrome c Group/chemistry , Cytochromes c , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Magnetic Resonance Spectroscopy , Models, Chemical , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Protein Structure, Secondary , Software , Solutions
7.
Proc Natl Acad Sci U S A ; 93(25): 14396-400, 1996 Dec 10.
Article in English | MEDLINE | ID: mdl-8962062

ABSTRACT

The solution structure of the three-heme electron transfer protein cytochrome c7 from Desulfuromonas acetoxidans is reported. The determination of the structure is obtained through NMR spectroscopy on the fully oxidized, paramagnetic form. The richness of structural motifs and the presence of three prosthetic groups in a protein of 68 residues is discussed in comparison with the four-heme cytochromes c3 already characterized through x-ray crystallography. In particular, the orientation of the three hemes present in cytochrome c7 is similar to that of three out of four hemes of cytochromes c3. The reduction potentials of the individual hemes, which have been obtained through the sequence-specific assignment of the heme resonances, are discussed with respect to the properties of the protein matrix. This information is relevant for any attempt to understand the electron transfer pathway.


Subject(s)
Cytochrome c Group/metabolism , Desulfovibrio/metabolism , Amino Acid Sequence , Cytochrome c Group/chemistry , Desulfovibrio/chemistry , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Oxidation-Reduction , Protein Conformation
8.
Biochemistry ; 34(36): 11385-98, 1995 Sep 12.
Article in English | MEDLINE | ID: mdl-7547866

ABSTRACT

The 1H NMR spectrum of the the cyanide adduct of a triply mutated Saccharomyces cerevisiae iso-1-cytochrome c (His39Gln/Met80Ala/Cys102Ser) in the oxidized form has been assigned through 1D NOE and 2D COSY, TOCSY, NOESY, and NOE-NOESY experiments; 562 protons out of a total of 683 have been assigned. The solution structure, the first of a paramagnetic heme protein, was determined using 1426 meaningful NOE constraints out of a total of 1842 measured NOEs. The RMSD values at the stage of restrained energy minimization of 17 structures obtained from distance geometry calculations are 0.68 +/- 0.11 and 1.32 +/- 0.14 A for the backbone and all heavy atoms, respectively. The quality, in terms of RMSD, of the present structure is the same as that obtained for the solution structure of the diamagnetic horse heart ferrocytochrome c [Qi, P. X., et al. (1994) Biochemistry 33, 6408-6419]. The secondary structure elements and the overall folding in the variant are observed to be the same as those of the wild-type protein for which the X-ray structure is available. However, the replacement of the methionine axial ligand with an alanine residue creates a ligand-binding "distal cavity". The properties of the distal cavity seen in this solution structure are compared to those of other heme proteins.


Subject(s)
Alanine/chemistry , Cyanides/chemistry , Cytochrome c Group/chemistry , Cytochromes c , Heme/chemistry , Methionine/chemistry , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Animals , Crystallography, X-Ray , Horses , Ligands , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Myocardium/enzymology , Protein Conformation , Protein Structure, Secondary , Solutions
9.
Biochemistry ; 34(31): 9851-8, 1995 Aug 08.
Article in English | MEDLINE | ID: mdl-7632685

ABSTRACT

The NMR solution structure of the oxidized HiPIP from Chromatium vinosum has been solved. Despite the fact that the protein is paramagnetic, 85% of the 1H and 80% of the 15N signals have been assigned. Through 1537 NOEs, out of which 1142 were found to be relevant for the structure determination, a family of structures has been obtained by distance geometry calculations. These structures have then been subjected to restrained energy minimization (REM) and restrained molecular dynamics (RMD) calculations in vacuum. Finally, the mean structure of the RMD family has been treated through RMD in water. The RMSD values for the backbone and heavy atoms within the RMD family are 0.57 +/- 0.14 and 1.08 +/- 0.16 A, respectively. These values together with other parameters indicate that the structure is of good quality and as good as the structure of the reduced protein. The RMDw structures of the reduced and oxidized proteins are different beyond the experimental indetermination. The set of constraints for the reduced and oxidized forms have been used to treat the available X-ray structure by RMD in water. The two structures generated in this way are quite similar to their respective solution structures, thus confirming that the experimental constraints are capable of yielding two different structures from the same starting structural model. This is the first time that independently determined solution structures of two redox states of a paramagnetic protein are available. Differences between them and the X-ray structure are discussed.


Subject(s)
Bacterial Proteins/chemistry , Chromatium/chemistry , Iron-Sulfur Proteins/chemistry , Photosynthetic Reaction Center Complex Proteins , Amino Acid Sequence , Computer Simulation , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Protein Conformation , Solutions
10.
Biochemistry ; 34(1): 206-19, 1995 Jan 10.
Article in English | MEDLINE | ID: mdl-7819198

ABSTRACT

The 1H NMR assignment of the reduced HiPIP from Chromatium vinosum available in the literature [Gaillard, J., Albrand, J.-P., Moulis, J.-M., & Wemmer, D. E. (1992) Biochemistry 31, 5632-5639] has been extended up to 85% of the total protein protons. Ninety percent of the nitrogens have been assigned. Then the solution structure has been obtained using as many as 1147 meaningful NOE connectivities. The protein is sizably paramagnetic even though the ground state is a singlet. Nevertheless, the final RMSD values are 0.62 and 1.19 A for the backbone and the heavy atoms, respectively. These values compare well with those for diamagnetic proteins of the same size. The solution structure is discussed in the light of the available structural information from X-ray data.


Subject(s)
Bacterial Proteins/chemistry , Chromatium/chemistry , Iron-Sulfur Proteins/chemistry , Photosynthetic Reaction Center Complex Proteins , Amino Acid Sequence , Crystallography, X-Ray , Hydrogen , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Nitrogen Isotopes , Oxidation-Reduction , Protein Conformation , Solutions
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