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3.
Curr Opin Struct Biol ; 11(2): 201-7, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11297928

ABSTRACT

Fluorescence resonance energy transfer (FRET) provides information on the distance between a donor and an acceptor dye in the range 10 to 100 A. Knowledge of the exact positions of some dyes with respect to nucleic acids now enables us to translate these data into precise structural information using molecular modeling. Advances in the preparation of dye-labeled nucleic acid molecules and in new techniques, such as the measurement of FRET in polyacrylamide gels or in vivo, will lead to an increasingly important role of FRET in structural and molecular biology.


Subject(s)
Biophysics/methods , DNA/metabolism , Energy Transfer , Escherichia coli Proteins , Proteins/metabolism , Spectrometry, Fluorescence/methods , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Biophysics/trends , Cyclic AMP Receptor Protein/chemistry , Cyclic AMP Receptor Protein/metabolism , DNA/chemistry , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/metabolism , Electrophoresis, Polyacrylamide Gel/methods , Fluorescent Dyes/chemistry , Integration Host Factors , Menotropins/chemistry , Menotropins/metabolism , Proteins/chemistry , Rec A Recombinases/chemistry , Rec A Recombinases/metabolism , Repressor Proteins/chemistry , Repressor Proteins/metabolism , TATA-Box Binding Protein , Transcription Factors/chemistry , Transcription Factors/metabolism
4.
Pharmazie ; 56(11): 843-9, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11817166

ABSTRACT

To improve the ratio of non-hormonal to hormonal activity, estrogens 3 and 4 were modified at various molecule positions. Isomerization of the 14 alpha,15 alpha-methylene bridge, controlled 3-methoxy group cleavage with respect to the 14 alpha,15 alpha-methylene bridge stereochemistry, reduction of the 8-double bond, and substitution of the oxyfunctionality at C-17 by a methylene and a difluoromethylene moiety were in the focus. As a result of in vivo and in vitro tests, compounds 27 and 29 were selected as potential follow-up candidates of lead 3.


Subject(s)
Estradiol Congeners/chemical synthesis , Estradiol/analogs & derivatives , Antioxidants/chemistry , Cyclopropanes/chemistry , Estradiol/chemical synthesis , Indicators and Reagents , Isomerism , Magnetic Resonance Spectroscopy , Molecular Conformation
5.
Anal Biochem ; 286(1): 26-34, 2000 Nov 01.
Article in English | MEDLINE | ID: mdl-11038269

ABSTRACT

Proteases play a key role in cellular biology and have become priority targets for new pharmaceuticals. Thus, there is a high demand for specific, sensitive, and quick assays to monitor the activity of endoproteases. We designed affinity-tagged helical proteins with unique protease cleavage sites and thus constructed universal, molecularly defined, and uniform substrates for in vitro detection of IgA endoprotease. The substrate is a 10.5-kDa recombinant helical protein with a high-affinity (His)(6)-tag at the amino-terminal end. Further elements are a unique proteolytic recognition site and a C-terminal helical extension, which is cut off by the protease. Proteolytic action can be monitored in real time using surface plasmon resonance spectroscopy. Femtomole amounts of protease could be reliably and quantitatively detected within a few minutes after the start of the reaction. The detection signal changed linearly with the amount of protease and was independent of the applied sample flow rate. The biochip can be reversibly loaded with the recombinant protease substrate, so that the SPR assay is well-suited for automation. By substituting an HIV protease site for the recognition site of the IgAse, we also obtained a substrate for the quantitative and sensitive detection of HIV-1 endoprotease. Our substrate design is thus generally applicable.


Subject(s)
Clinical Chemistry Tests/methods , Endopeptidases/analysis , Endopeptidases/metabolism , Surface Plasmon Resonance/methods , Amino Acid Sequence , Base Sequence , Binding Sites , Dose-Response Relationship, Drug , Endopeptidases/chemistry , Fluorescent Dyes/metabolism , HIV Protease/analysis , HIV Protease/chemistry , Molecular Sequence Data , Mutagenesis, Insertional , Recombinant Proteins/metabolism , Serine Endopeptidases/analysis , Serine Endopeptidases/chemistry , Time Factors
6.
Proteins ; 39(4): 408-16, 2000 Jun 01.
Article in English | MEDLINE | ID: mdl-10813822

ABSTRACT

Plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number. It acts as a transcriptional repressor at the essential repR promoter. Furthermore, CopR prevents convergent transcription from the repR and the antisense promoter, thereby indirectly increasing the amount of antisense-RNA, the second regulatory component. CopR binds as a dimer to a nearly palindromic operator with the consensus sequence 5'CGTG. Previously, a CopR structural model was built and used to identify amino acids involved in DNA binding. These data showed that CopR is a HTH protein belonging to the lambda repressor superfamily and allowed the identification of two amino acids involved in specific DNA recognition. Here, we describe site-directed mutagenesis in combination with EMSA, dimerization studies using sedimentation equilibrium, and CD measurements to verify the model predictions concerning amino acids involved in dimerization. With this approach, the dimeric interface could be located between amino acids I44 and L62. F5 located at the N-terminus is additionally required for proper folding, and could, therefore, not be unequivocally assigned to the dimeric interface. CD measurements at protein concentrations well below K(Dimer) revealed that the monomer of CopR is folded.


Subject(s)
Bacterial Proteins , DNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , Trans-Activators/metabolism , Amino Acid Sequence , Amino Acids , Bacillus subtilis , Circular Dichroism , DNA/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Dimerization , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Secondary , Repressor Proteins/chemistry , Repressor Proteins/genetics , Trans-Activators/chemistry , Trans-Activators/genetics , Transcription, Genetic
7.
Proteins ; 38(4): 393-406, 2000 Mar 01.
Article in English | MEDLINE | ID: mdl-10707026

ABSTRACT

The plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number. CopR binds as a dimer to a nearly palindromic operator with the consensus sequence 5'-CGTG. Intermediate sequence searches revealed a significant structural relationship between CopR and the bacteriophage P22 c2 and the 434 c1 repressors. In this report we describe the experimental verification of a CopR homology model, which is based on a fairly low-sequence identity of 13.8% to P22 c2 repressor. A model for the complex of CopR with the deoxyribonucleic acid (DNA) target was built on the basis of experimental footprinting data, the above-mentioned CopR homology model, and the crystal structure of the 434 c1 repressor-DNA complex. Site-directed mutagenesis was used to test the function of amino acids involved in sequence and nonsequence-specific DNA recognition and amino acids important for correct protein folding. CD measurements were performed to detect structural changes caused by the mutations. Exchanges of residues responsible for sequence-specific DNA recognition reduced binding to a nonspecific level. Mutations of amino acids involved in nonspecific DNA binding lead to decreased binding affinity while maintaining selectivity. Substitution of amino acids necessary for proper folding caused dramatic structural changes. The experimental data support the model of CopR as a helix-turn-helix protein belonging to the lambda repressor superfamily.


Subject(s)
Bacterial Proteins , DNA-Binding Proteins/chemistry , Escherichia coli Proteins , Repressor Proteins/chemistry , Trans-Activators/chemistry , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution , Bacillus subtilis/metabolism , Base Sequence , Circular Dichroism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Protein Structure, Tertiary , Repressor Proteins/genetics , Repressor Proteins/metabolism , Sequence Alignment , Sequence Deletion , Trans-Activators/genetics , Trans-Activators/metabolism
8.
J Mol Biol ; 302(5): 1081-100, 2000 Oct 06.
Article in English | MEDLINE | ID: mdl-11183776

ABSTRACT

DNA molecules with three bulges separated by double-stranded helical sections of B-DNA were constructed to be used as substrates for DNA-protein binding assays. Fluorescence resonance energy transfer (FRET) between dye molecules attached to the 5'-ends of the DNA molecules is used to monitor the protein binding. The A5 bulge, which consists of five unpaired adenine nucleotides, alters the direction of the helical axis by approximately 80 to 90 at every bulge site. Computer molecular modeling facilitated a pre-selection of suitable helix lengths that bring the labeled ends of the three-bulge DNA molecules (60 to 70 base-pairs long) into close proximity. The FRET experiments verified that the labeled ends of the helices of these long molecules were indeed close. A series of FRET experiments was carried out with two A5 and two A7 bulge molecules. The relative positions of the bulges were varied along the central helical DNA sequence (between the bulges) in order to determine the relative angular juxtapositions of the outlying helical arms flanking the central helical region. The global structural features of the DNA molecules are manifested in the FRET data. The FRET experiments, especially those of the two-bulge series, could be interpreted remarkably well with molecular models based on the NMR structure of the A5 bulge. These models assume that the DNA molecules do not undergo large torsional conformational fluctuations at the bulge sites. The magnitude of the FRET efficiency attests to a relatively rigid structure for many of the long 5'-end-labeled molecules. The changes in the FRET efficiency of three-bulge structures containing the specific binding sequence of the catabolite activator protein (CAP) demonstrated significant deformation of the DNA upon binding of CAP. No direct interaction of CAP with the dyes was observed.


Subject(s)
Base Pair Mismatch , Cyclic AMP Receptor Protein/metabolism , DNA-Binding Proteins/metabolism , DNA/chemistry , DNA/metabolism , Nucleic Acid Conformation , Adenine/metabolism , Base Pair Mismatch/genetics , Base Pairing , Base Sequence , Computer Simulation , DNA/genetics , Energy Transfer , Fluorescein/metabolism , Fluorescence Polarization , Fluorescent Dyes/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Nucleic Acid Denaturation , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/metabolism , Rhodamines/metabolism , Temperature , Thermodynamics , Titrimetry
9.
J Mol Biol ; 294(1): 79-91, 1999 Nov 19.
Article in English | MEDLINE | ID: mdl-10556030

ABSTRACT

The high mobility group (HMG) domain is a DNA binding motif found in some eukaryotic chromosomal proteins and transcription factors. This domain binds in the minor groove of DNA inducing a sharp bend and also preferentially binds to certain distorted DNA structures. Although structures of sequence-specific HMG domains with their cognate double-helical DNA binding sites have been solved, the nature of the interaction of the domain with distorted DNA remains to be established. In this study we have investigated the interaction of HMG-D, a Drosophila counterpart of the vertebrate HMG1, with a DNA oligomer containing a bulge of two adenine residues. We show by footprinting that HMG-D binds preferentially on one side of the bulged DNA. Based on these data and on the published NMR structures of the HMG domain of HMG-D and the LEF-1-DNA complex, we modelled the HMG-D - bulged DNA complex. This model predicts that two residues, Val32 and Thr33, in the loop between alpha-helices I and II are inserted deep into the "hole" in the DNA formed by the two missing bases on one strand of the DNA bulge. Mutation of these residues confirmed that both are required for the efficient binding and bending of DNA by HMG-D. We discuss both the role of this loop in the recognition of distorted DNA structures by non-sequence specific HMG domain proteins and that of the basic tail in stabilising the induced DNA bend.


Subject(s)
DNA-Binding Proteins/metabolism , DNA/chemistry , DNA/metabolism , High Mobility Group Proteins/metabolism , Amino Acid Sequence , Binding Sites , Conserved Sequence , DNA Footprinting , DNA, Circular/metabolism , Hydroxyl Radical , Lymphoid Enhancer-Binding Factor 1 , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Transcription Factors/metabolism
10.
Nucleic Acids Res ; 27(23): 4619-25, 1999 Dec 01.
Article in English | MEDLINE | ID: mdl-10556318

ABSTRACT

We have analyzed the structure of two related protein-DNA complexes consisting of integration host factor (IHF) bound to two different versions of the H' site of bacteriophage lambda. Both DNA substrates were 55 bp in length. While one was native duplex the other possessed a nick in one strand at a crucial position within the IHF consensus at the same position as in the reported crystal structure of the DNA-IHF complex. By labeling the 5'-ends of these DNA molecules with donor and acceptor fluorescent dyes, we were able to measure the distance between the dyes by fluorescence resonance energy transfer (FRET) and model DNA distortion. The FRET efficiency decreased from 0.49 +/- 0.01 (nicked DNA) to 0.37 +/- 0.01 (intact DNA) when the gap in the DNA strand was closed. The measured dye-to-dye distance of IHF in complex with nicked DNA was in agreement with the expected value from the crystal structure. Although we found that the two structures were distinguishable, the global shape induced by IHF was retained between the two DNA molecules. Furthermore, our FRET and modeling techniques have sufficiently high resolution to distinguish subtle changes in nucleoprotein complexes with biological relevance.


Subject(s)
Bacterial Proteins/chemistry , DNA/chemistry , Nucleic Acid Conformation , Base Sequence , Electrophoresis, Polyacrylamide Gel , Integration Host Factors , Models, Chemical , Molecular Sequence Data , Spectrometry, Fluorescence
11.
Biochemistry ; 38(39): 12860-8, 1999 Sep 28.
Article in English | MEDLINE | ID: mdl-10504256

ABSTRACT

The three-dimensional solution structure of a DNA molecule of the sequence 5'-d(GCATCGAAAAAGCTACG)-3' paired with 5'-d(CGTAGCCGATGC)-3' containing a five-adenine bulge loop (dA(5)-bulge) between two double helical stems was determined by 2D (1)H and (31)P NMR, infrared, and Raman spectroscopy. The DNA in both stems adopt a classical B-form double helical structure with Watson-Crick base pairing and C2'-endo sugar conformation. In addition, the two dG/dC base pairs framing the dA(5)-bulge loop are formed and are stable at least up to 30 degrees C. The five adenine bases of the bulge loop are localized at intrahelical positions within the double helical stems. Stacking on the double helical stem is continued for the first four 5'-adenines in the bulge loop. The total rise (the height) of these four stacked adenines roughly equals the diameter of the double helical stem. The stacking interactions are broken between the last of these four 5'-adenines and the fifth loop adenine at the 3'-end. This 3'-adenine partially stacks on the other stem. The angle between the base planes of the two nonstacking adenines (A10 and A11) in the bulge loop reflects the kinking angle of the global DNA structure. The neighboring cytosines opposite the dA(5)-bulge (being parts of the bulge flanking base pairs) do not stack on one another. This disruption of stacking is characterized by a partial shearing of these bases, such that certain sequential NOEs for this base step are preserved. In the base step opposite the loop, an extraordinary hydrogen bond is observed between the phosphate backbone of the 5'-dC and the amino proton of the 3'-dC in about two-thirds of the conformers. This hydrogen bond probably contributes to stabilizing the global DNA structure. The dA(5)-bulge induces a local kink into the DNA molecule of about 73 degrees (+/-11 degrees ). This kinking angle and the mutual orientation of the two double helical stems agree well with results from fluorescence resonance energy transfer measurements of single- and double-bulge DNA molecules.


Subject(s)
Adenine/chemistry , DNA/chemistry , Nucleic Acid Conformation , Base Sequence , Energy Transfer , Fluorescence , Magnetic Resonance Spectroscopy , Models, Molecular , Solutions
12.
Biochemistry ; 38(37): 12150-8, 1999 Sep 14.
Article in English | MEDLINE | ID: mdl-10508419

ABSTRACT

The HMG domains of the chromosomal high mobility group proteins homologous to the vertebrate HMG1 and HMG2 proteins preferentially recognize distorted DNA structures. DNA binding also induces a substantial bend. Using fluorescence resonance energy transfer (FRET), we have determined the changes in the end-to-end distance consequent on the binding of selected insect counterparts of HMG1 to two DNA fragments, one of 18 bp containing a single dA(2) bulge and a second of 27 bp with two dA(2) bulges. The observed changes are consistent with overall bend angles for the complex of the single HMG domain with one bulge and of two domains with two bulges of approximately 90-100 degrees and approximately 180-200 degrees, respectively. The former value contrasts with an inferred value of 150 degrees reported by Heyduk et al. (1) for the bend induced by a single domain. We also observe that the induced bend angle is unaffected by the presence of the C-terminal acidic region. The DNA bend of approximately 95 degrees observed in the HMG domain complexes is similar in magnitude to that induced by the TATA-binding protein (80 degrees), each monomeric unit of the integration host factor (80 degrees), and the LEF-1 HMG domain (107 degrees). We suggest this value may represent a steric limitation on the extent of DNA bending induced by a single DNA-binding motif.


Subject(s)
DNA/chemistry , DNA/metabolism , High Mobility Group Proteins/chemistry , High Mobility Group Proteins/metabolism , Nucleic Acid Conformation , Amino Acid Sequence , Animals , Chironomidae , DNA, Circular/chemistry , DNA, Circular/metabolism , Drosophila , Energy Transfer , Fluoresceins/chemistry , Fluorescent Dyes/chemistry , Molecular Sequence Data , Protein Binding , Rhodamines/chemistry , Saccharomyces cerevisiae , Spectrometry, Fluorescence/methods
13.
Steroids ; 63(1): 21-7, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9437791

ABSTRACT

The 5 alpha- and 5 beta-oriented delta 3-double bond isomers 8, 9 of the widely used progestin desogestrel (7) were synthesized. Wittig olefination reaction of the 5 alpha-intermediate 12 showed a dramatically reduced reaction rate compared with the olefination of the 5 beta-intermediate 13. Computational studies suggest that different energies of the intermediary 1,2-oxaphosphetanes may, at least partially, have been the reason for this phenomenon.


Subject(s)
Alkenes/chemistry , Desogestrel/chemical synthesis , Crystallization , Crystallography, X-Ray , Desogestrel/chemistry , Mathematical Computing , Stereoisomerism
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