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1.
J Am Chem Soc ; 123(48): 12009-17, 2001 Dec 05.
Article in English | MEDLINE | ID: mdl-11724609

ABSTRACT

Experimental and ab initio molecular orbital techniques are developed for study of aluminum species with large quadrupole coupling constants to test structural models for methylaluminoxanes (MAO). The techniques are applied to nitrogen- and oxygen-containing complexes of aluminum and to solid MAO isolated from active commercial MAO preparations. (Aminato)- and (propanolato)aluminum clusters with 3-, 4-, and 6-coordinate aluminum sites are studied with three (27)Al NMR techniques optimized for large (27)Al quadrupole coupling constants: field-swept, frequency-stepped, and high-field MAS NMR. Four-membered (aminato)aluminum complexes with AlN(4) coordination yield slightly smaller C(q) values than similar AlN(2)C(2) sites: 12.2 vs 15.8 MHz. Planar 3-coordinate AlN(2)C sites have the largest C(q) values, 37 MHz. In all cases, molecular orbital calculations of the electric field gradient tensors yields C(q) and eta values that match with experiment, even for a large hexameric (aminato)aluminum cage. A D(3d) symmetry hexaaluminum oxane cluster, postulated as a model for MAO, yields a calculated C(q) of -23.7 MHz, eta = 0.7474, and predicts a spectrum that is too broad to match the field-swept NMR of methylaluminoxane, which shows at least three sites, all with C(q) values greater than 15 MHz but less than 21 MHz. Thus, the proposed hexaaluminum cluster, with its strained four-membered rings, is not a major component of MAO. However, calculations for dimers of the cage complex, either edge-bridged or face-bridged, show a much closer match to experiment. Also, MAO preparations differ, with a gel form of MAO having significantly larger (27)Al C(q) values than a nongel form, a conclusion reached on the basis of (27)Al NMR line widths in field-swept NMR spectra acquired from 13 to 24 T.

2.
Nat Struct Biol ; 8(10): 833-7, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11573085

ABSTRACT

The RING domain of the breast and ovarian cancer tumor suppressor BRCA1 interacts with multiple cognate proteins, including the RING protein BARD1. Proper function of the BRCA1 RING domain is critical, as evidenced by the many cancer-predisposing mutations found within this domain. We present the solution structure of the heterodimer formed between the RING domains of BRCA1 and BARD1. Comparison with the RING homodimer of the V(D)J recombination-activating protein RAG1 reveals the structural diversity of complexes formed by interactions between different RING domains. The BRCA1-BARD1 structure provides a model for its ubiquitin ligase activity, illustrates how the BRCA1 RING domain can be involved in associations with multiple protein partners and provides a framework for understanding cancer-causing mutations at the molecular level.


Subject(s)
BRCA1 Protein/chemistry , Carrier Proteins/chemistry , Tumor Suppressor Proteins , Ubiquitin-Protein Ligases , Amino Acid Sequence , BRCA1 Protein/genetics , Breast Neoplasms/genetics , Dimerization , Female , Genes, Tumor Suppressor , Genetic Predisposition to Disease , Humans , Molecular Sequence Data , Mutation , Ovarian Neoplasms/genetics , Protein Conformation , Sequence Homology, Amino Acid
3.
Nat Struct Biol ; 8(4): 349-52, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11276257

ABSTRACT

BAG-family proteins share a conserved protein interaction region, called the 'BAG domain', which binds and regulates Hsp70/Hsc70 molecular chaperones. This family of cochaperones functionally regulates signal transducing proteins and transcription factors important for cell stress responses, apoptosis, proliferation, cell migration and hormone action. Aberrant overexpression of the founding member of this family, BAG1, occurs in human cancers. In this study, a structure-based approach was used to identify interacting residues in a BAG1--Hsc70 complex. An Hsc70-binding fragment of BAG1 was shown by multidimensional NMR methods to consist of an antiparallel three-helix bundle. NMR chemical shift experiments marked surface residues on the second (alpha 2) and third (alpha 3) helices in the BAG domain that are involved in chaperone binding. Structural predictions were confirmed by site-directed mutagenesis of these residues, resulting in loss of binding of BAG1 to Hsc70 in vitro and in cells. Molecular docking of BAG1 to Hsc70 and mutagenesis of Hsc70 marked the molecular surface of the ATPase domain necessary for interaction with BAG1. The results provide a structural basis for understanding the mechanism by which BAG proteins link molecular chaperones and cell signaling pathways.


Subject(s)
HSP70 Heat-Shock Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Computer Simulation , DNA-Binding Proteins , Genes, Reporter , HSC70 Heat-Shock Proteins , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/genetics , Membrane Proteins/chemistry , Mice , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Tertiary , Qa-SNARE Proteins , Receptors, Androgen/metabolism , Sequence Alignment , Transcription Factors/genetics , Transcriptional Activation
4.
J Mol Biol ; 303(4): 555-65, 2000 Nov 03.
Article in English | MEDLINE | ID: mdl-11054291

ABSTRACT

Transthyretin is a human protein capable of amyloid formation that is believed to cause several types of amyloid disease, depending on the sequence deposited. Previous studies have demonstrated that wild-type transthyretin (TTR), although quite stable, forms amyloid upon dissociation from its native tetrameric form into monomers with an altered conformation. Many naturally occurring single-site variants of TTR display decreased stability in vitro, manifested by the early onset familial amyloid diseases in vivo. Only subtle structural changes were observed in X-ray crystallographic structures of these disease associated variants. In this study, the stability of the wild-type TTR tetramer was investigated at the residue-resolution level by monitoring (2)H-H exchange via NMR spectroscopy. The measured protection factors for slowly-exchanging amide hydrogen atoms reveal a stable core consisting of strands A, B, E, F, and interestingly, the loop between strands A and B. In addition, the faster exchange of amide groups from residues at the subunit interfaces suggests unexpected mobility in these regions. This information is crucial for future comparisons between disease-associated and wild-type tetramers. Such studies can directly address the regions of TTR that become destabilized as a consequence of single amino acid substitutions, providing clues to aspects of TTR amyloidogenesis.


Subject(s)
Deuterium/metabolism , Prealbumin/chemistry , Prealbumin/metabolism , Amides/metabolism , Binding Sites , Dimerization , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Quaternary , Protein Subunits , Protons
5.
Biochim Biophys Acta ; 1392(1): 101-8, 1998 May 20.
Article in English | MEDLINE | ID: mdl-9593844

ABSTRACT

Pulsed-field-gradient NMR spectroscopy was used to measure translational diffusion coefficients (Ds) for a peptide corresponding to a proposed lipid-binding domain of human apolipoprotein C-I, residues 7-24 (apoC-I(7-24)). Diffusion coefficients for apoC-I(7-24) were determined directly by following the decay of the resonance intensity of selected peptide protons at various concentrations of sodium dodecyl sulfate (SDS), a detergent increasingly being used to model the apolipoprotein environment. Previously, diffusion coefficients of peptides in the presence of SDS have been determined indirectly by monitoring the SDS diffusion coefficient. The direct measurement of the diffusion coefficient of the peptide enables one to distinguish whether SDS simply coats the peptide's surface to produce a uniformly charged 'rod' or if the peptide associates with a micelle. Using the direct method, at SDS concentrations above 5 mM (which is below the SDS critical micelle concentration (8.1 mM)), apoC-I(7-24) exhibited diffusion coefficients consistent with the formation of a large-molecular-weight complex. Based on the ratio of the diffusion coefficients for free- and SDS-associated peptide, the molecular weight of the peptide-SDS complex was much larger than a factor of 1. 4, the increase in molecular weight of the free peptide predicted if apoC-I(7-24) was uniformly surface coated with SDS.


Subject(s)
Apolipoproteins C/chemistry , Apolipoprotein C-I , Binding Sites , Diffusion , Micelles , Nuclear Magnetic Resonance, Biomolecular/methods , Peptide Fragments/chemistry , Sodium Dodecyl Sulfate/chemistry
6.
J Biochem ; 122(4): 686-90, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9399568

ABSTRACT

TGF-alpha, a 50 amino acid growth factor containing 3 disulfide bonds, was fused to the N-terminal domain of the pIII protein of fusN, a derivative of phagemid fd-tet, to form a TGF-alpha phage. The fusion phage showed binding activity to epidermal growth factor receptor (EGFR). A library of approximately 4 x 10(7) variants of TGF-alpha was generated with substitutions of total of 10 amino acids located in the C-loop region. This C-loop subdomain of TGF-alpha consists of a small antiparallel double hairpin structure involving interactions between intra-polypeptide segments. Mutants isolated from the phage library with greatly increased binding affinity were selected through panning with A431 cells (a cell line expressing an elevated number of EGFRs). Following two rounds of stringent selection, variant phages with higher binding affinity than wild type TGF-alpha were identified and the phage DNAs were sequenced for the alignment analysis. Absolute selection at position 42 as Arg, preferential selection at position 38 and 45 as Tyr or Phe with aromatic side chain and selection at position 41 with acidic residues, were obtained. Although an amino acid residue with smaller side chain at position 35 and one with larger side chain at position 36 were preferred, the steric hindering of the structure in side chains was minimized between these adjacent amino acids.


Subject(s)
Bacteriophages/genetics , ErbB Receptors/metabolism , Transforming Growth Factor alpha/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cloning, Molecular , Humans , Mutagenesis , Plasmids , Protein Binding , Sequence Homology, Amino Acid , Transforming Growth Factor alpha/metabolism
7.
J Biol Chem ; 271(50): 32204-11, 1996 Dec 13.
Article in English | MEDLINE | ID: mdl-8943277

ABSTRACT

The study of human transforming growth factor-alpha (TGF-alpha) in complex with the epidermal growth factor (EGF) receptor extracellular domain has been undertaken in order to generate information on the interactions of these molecules. Analysis of 1H NMR transferred nuclear Overhauser enhancement data for titration of the ligand with the receptor has yielded specific data on the residues of the growth factor involved in contact with the larger protein. Significant increases and decreases in nuclear Overhauser enhancement cross-peak intensity occur upon complexation, and interpretation of these changes indicates that residues of the A- and C-loops of TGF-alpha form the major binding interface, while the B-loop provides a structural scaffold for this site. These results corroborate the conclusions from NMR relaxation studies (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283-15292), which suggest that the C-terminal residues of the polypeptide are immobilized upon receptor binding, while the N terminus of the molecule retains considerable flexibility, and are consistent with structure-function studies of the TGF-alpha/EGF system indicating a multidomain binding model. These results give a visualization, for the first time, of native TGF-alpha in complex with the EGF receptor and generate a picture of the ligand-binding site based upon the intact molecule. This will undoubtedly be of utility in the structure-based design of TGF-alpha/EGF agonists and/or antagonists.


Subject(s)
ErbB Receptors/metabolism , Transforming Growth Factor alpha/metabolism , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Conformation
8.
Biochemistry ; 33(51): 15283-92, 1994 Dec 27.
Article in English | MEDLINE | ID: mdl-7803391

ABSTRACT

The interaction of transforming growth factor alpha (TGF-alpha) with the complete extracellular domain of the epidermal growth factor receptor (EGFR-ED) was examined by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR resonances of the methyl groups of TGF-alpha were used as probes of the interaction of TGF-alpha with the EGF receptor to determine the binding kinetics and the differential mobility within the bound TGF-alpha. The methyl resonances were studied because there are 14 methyl containing residues well dispersed throughout the structure of TGF-alpha and the relaxation properties of methyl groups are well understood. Changes in the longitudinal and transverse 1H NMR relaxation rates of the methyl resonances of TGF-alpha caused by binding to the 85-kDa EGFR-ED were studied. From these measurements it was determined that the interaction was in the NMR fast exchange limit. A binding mechanism to rationalize the different rates determined by NMR and surface plasmon resonance techniques [Zhou, M., et al. (1993) Biochemistry 32, 8193-8198] is proposed. The transverse relaxation rate (R2) enhancements of the various methyl resonances displayed a regional dependence within the bound TGF-alpha molecule. Resonances from the C-terminus of TGF-alpha, which were flexible in the unbound molecule, revealed dramatic increases in their R2 upon binding to the EGFR-ED along with resonances from the interior of TGF-alpha. However, upon binding, the R2 enhancements of the methyl resonances from the N-terminus of TGF-alpha, which were also flexible in the unbound TGF-alpha, were slight; indicating a retention of mobility of this region for bound TGF-alpha. The implications of these data with respect to the mechanism of receptor activation and the design of antagonists are discussed.


Subject(s)
ErbB Receptors/chemistry , Transforming Growth Factor alpha/chemistry , Amino Acid Sequence , In Vitro Techniques , Kinetics , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Motion , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins , Solutions
9.
Biochim Biophys Acta ; 1159(1): 81-93, 1992 Sep 04.
Article in English | MEDLINE | ID: mdl-1390913

ABSTRACT

Previous studies on mitochondrial targeting presequences have indicated that formation of an amphiphillic helix may be required for efficient targeting of the precursor protein into mitochondria, but the structural details are not well understood. We have used CD and NMR spectroscopy to characterize in detail the structure of a synthetic peptide corresponding to the presequence for the beta-subunit of F1-ATPase, a mitochondrial matrix protein. Although this peptide is essentially unstructured in water, alpha-helix formation is induced when the peptide is placed in structure-promoting environments, such as SDS micelles or aqueous trifluoroethanol (TFE). In 50% TFE (by volume), the peptide is in dynamic equilibrium between random coil and alpha-helical conformations, with a significant population of alpha-helix throughout the entire peptide. The helix is somewhat more stable in the N-terminal part of the presequence (residues 4-10), and this result is consistent with the structure proposed previously for the presequence of another mitochondrial matrix protein, yeast cytochrome oxidase subunit IV. Addition of increasing amounts of TFE causes the alpha-helical content to increase even further, and the TFE titration data for the presequence peptide of the F1-ATPase beta-subunit are not consistent with a single, cooperative transition from random coil to alpha-helix. There is evidence that helix formation is initiated in two different regions of the peptide. This result helps to explain the redundancy of the targeting information contained in the presequence for the F1-ATPase beta-subunit.


Subject(s)
Proton-Translocating ATPases/chemistry , Amino Acid Sequence , Biological Transport , Circular Dichroism , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Mitochondria/metabolism , Molecular Sequence Data , Peptides/chemistry , Protein Precursors/chemistry , Protein Structure, Secondary
10.
J Biol Chem ; 266(32): 21693-9, 1991 Nov 15.
Article in English | MEDLINE | ID: mdl-1834660

ABSTRACT

The transport of the F1-ATPase beta-subunit precursor into mitochondria is dependent upon a presequence at its amino terminus. Within the mitochondrial membrane translocation site the potential amphiphilic character of the presequence region may be necessary to stabilize binding to the mitochondrial inner membrane. To better understand its role in protein import, the interaction of the F1 beta-presequence with lipid membranes was measured using circular dichroism and surface tensiometry. These studies reveal that a 20-residue peptide containing the F1 beta-presequence binds to phospholipid vesicles (Kd = 4.5-6.0 x 10(-8)M and adopts a predominantly alpha-helical structure. Although the presequence peptide binds avidly to lipids, it does not appear to penetrate deeply into the bilayer to perturb a reporter probe in the membrane interior. Compared with the effect of the peptides with demonstrated membrane insertion and lytic properties, the F1-beta-presequence appears to displace phospholipid head groups but not insert deeply into the bilayer. High concentrations (greater than 50 microM) of presequence peptides are required to noticibly perturb import of the full length F1 alpha- or F1 beta-subunit precursors. Thus, the F1 beta-presequence alone is not sufficient to efficiently compete for import but may require a protein context or a minimal length to assist insertion into the transport site. These observations are discussed in light of the different requirements for import of various presequence containing precursors into mitochondria.


Subject(s)
Enzyme Precursors/metabolism , Intracellular Membranes/metabolism , Mitochondria/metabolism , Peptides/metabolism , Proton-Translocating ATPases/metabolism , Submitochondrial Particles/metabolism , Amino Acid Sequence , Animals , Circular Dichroism , Enzyme Precursors/genetics , Escherichia coli/genetics , Fluorescence Polarization , Kinetics , Lipid Bilayers , Molecular Sequence Data , Oxygen Consumption , Peptides/chemical synthesis , Protein Binding , Protein Biosynthesis , Protein Conformation , Protein Sorting Signals/genetics , Protein Sorting Signals/metabolism , Proton-Translocating ATPases/genetics , Restriction Mapping , Surface Tension , Transcription, Genetic
11.
Biochemistry ; 30(42): 10155-63, 1991 Oct 22.
Article in English | MEDLINE | ID: mdl-1931946

ABSTRACT

Peptides corresponding to the wild-type signal sequence of the Escherichia coli outer membrane protein OmpA and several mutants have been synthesized and characterized biophysically. The mutations were designed collaboratively with Inouye and co-workers to test the understanding of the critical characteristics of signal sequences required for their functions. The in vivo results for these mutants have been reported [Lehnhardt, S., Pollitt, S., & Inouye, M. (1987) J. Biol. Chem. 262, 1716-1719; Goldstein, J., Lehnhardt, S., & Inouye, M. (1990) J. Bacteriol. 172, 1225-1231; Goldstein, J., Lehnhardt, S., & Inouye, M. (1991) J. Biol. Chem. 266, 14413-14417], and the present paper compares the conformational and membrane-interactive properties of six of the OmpA signal peptides. Peptides corresponding to functional OmpA signal sequences in vivo are predominantly alpha-helical in membrane-mimetic environments and insert readily into phospholipid bilayers. Nonfunctional OmpA signal peptides may have high helical content but do not penetrate deeply into the acyl chain region of bilayers. The ability of the signal peptides to insert into membranes and their in vivo function correlate with the residue-average hydrophobicity of their hydrophobic cores. The results obtained on OmpA signal peptides parallel closely our previous observations on peptides corresponding to the LamB signal sequence and mutants, arguing that the critical biophysical properties of signal sequences are general despite their lack of primary sequence identity.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Lipids/chemistry , Mutation , Protein Sorting Signals/genetics , Protein Sorting Signals/physiology , Amino Acid Sequence , Fluoresceins , Fluorescence Polarization , Micelles , Molecular Sequence Data , Protein Conformation , Solubility
12.
J Biol Chem ; 266(22): 14406-12, 1991 Aug 05.
Article in English | MEDLINE | ID: mdl-1860847

ABSTRACT

We have examined the comparative membrane interaction properties of synthetic peptides corresponding to the wild-type and an export-defective, mutated signal sequence from the Escherichia coli outer membrane protein, OmpA. As part of a collaborative study of the effects of various alterations on the function of the OmpA signal sequence and the biophysical properties of the corresponding synthetic peptides, we incorporated the small, neutral polar residue, asparagine, into the hydrophobic core in place of Ile-8. This seemingly minor perturbation to the signal sequence caused a complete block of export in vivo (J. Goldstein, S. Lehnhardt, and M. Inouye, following paper). We now explore in detail the difference in the properties of the wild-type and the Ile-8----Asn OmpA signal peptides. The fluorescent residue Trp was substituted in both peptides in place of the wild-type Phe at position 15. This mutation is silent phenotypically and provides a superb probe of membrane interaction. We find that the Asn substitution leaves the conformational properties of the signal sequence essentially unchanged, but prevents any significant interaction of the peptide with a lipid bilayer. Asparagines are very underrepresented among known signal sequences. We believe this low frequency to be due to the lowering of mean residue hydrophobicity caused by incorporation of Asn and the consequent reduced ability to bind and insert into membranes.


Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Escherichia coli/metabolism , Mutation , Protein Sorting Signals/genetics , Bacterial Outer Membrane Proteins/genetics , Circular Dichroism , Fluorescence Polarization , Lipid Bilayers , Protein Sorting Signals/metabolism , Tryptophan
13.
JAMA ; 258(4): 504-9, 1987.
Article in English | MEDLINE | ID: mdl-3599347

ABSTRACT

Urinalysis results are increasingly being used by employers to detect use of prohibited substances in the workplace. Actions taken on the basis of positive urinalysis results can have a significant effect on employees' lives and careers, and may face legal challenge. Important aspects of urinalysis are choice of analytical methods and their legal defensibility. Medical directors are frequently required to select testing laboratories, but have had few data correlating analytical methods with legal defensibility. To evaluate the legal defensibility of the methods used for drug testing, we conducted a survey of technical experts, testing laboratories, and arbitrators. Experts reported wide differences in the legal defensibility of methods rated. Arbitrators had considerable experience in drug use cases and understood the critical role of urinalysis results, but were unable to distinguish legal defensibility of analytical methods. Commercial laboratory practice in urinalysis varies widely among laboratories. An objective of this article is to provide company medical directors with information that will enable them to make informed decisions when choosing urinalysis laboratories and methods.


Subject(s)
Civil Rights/legislation & jurisprudence , Occupational Medicine , Substance-Related Disorders/diagnosis , Chemistry, Clinical/methods , Data Collection , Employment , Humans , Illicit Drugs/urine , Substance-Related Disorders/urine , United States
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