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1.
Proc Natl Acad Sci U S A ; 103(37): 13658-63, 2006 Sep 12.
Article in English | MEDLINE | ID: mdl-16954199

ABSTRACT

The fold of a helical membrane protein is largely determined by interactions between membrane-imbedded helices. To elucidate recurring helix-helix interaction motifs, we dissected the crystallographic structures of membrane proteins into a library of interacting helical pairs. The pairs were clustered according to their three-dimensional similarity (rmsd

Subject(s)
Membrane Proteins/chemistry , Amino Acid Motifs , Protein Folding , Protein Structure, Secondary
2.
J Pept Res ; 65(3): 312-21, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15787961

ABSTRACT

Water-soluble phospholamban (WSPLB) is a designed, water-soluble analogue of the pentameric membrane protein phospholamban (PLB), which contains the same core and interhelical residues as PLB, with only the solvent-exposed positions mutated. WSPLB contains the same secondary and quaternary structure as PLB. The hydrophobic cores of PLB and WSPLB contain Leu and Ile at the a- and d-positions of a heptad repeat (abcdefg) from residues 31-52, while residues 21-30 are rich in polar amino acids at these positions. While the full-length WSPLB forms pentamers in solution, truncated peptides lacking residues 21-30 are largely tetrameric. Thus, truncation of residues 1-20 promotes a switch from pentamer to tetramer formation. Here, the motifs for WSPLB pentamerization were elucidated by characterizing a series of peptides, which were progressively truncated in this polar 'switch' region. When fully present, the 'switch' region promotes pentamer formation in WSPLB, by destabilizing a more stable tetrameric species which exists in its absence. We find that the burial of hydrogen bonding residues from 21 to 30 drives WSPLB from a tetramer to a pentamer, with direct implications for coiled-coil design.


Subject(s)
Amino Acid Motifs , Calcium-Binding Proteins/chemistry , Protein Engineering , Circular Dichroism , Models, Molecular , Peptide Fragments/chemistry , Protein Denaturation , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Solubility , Water
3.
Proc Natl Acad Sci U S A ; 101(32): 11566-70, 2004 Aug 10.
Article in English | MEDLINE | ID: mdl-15292507

ABSTRACT

The de novo design of catalytic proteins provides a stringent test of our understanding of enzyme function, while simultaneously laying the groundwork for the design of novel catalysts. Here we describe the design of an O(2)-dependent phenol oxidase whose structure, sequence, and activity are designed from first principles. The protein catalyzes the two-electron oxidation of 4-aminophenol (k(cat)/K(M) = 1,500 M(-1).min(-1)) to the corresponding quinone monoimine by using a diiron cofactor. The catalytic efficiency is sensitive to changes of the size of a methyl group in the protein, illustrating the specificity of the design.


Subject(s)
Monophenol Monooxygenase/chemical synthesis , Protein Engineering/methods , Aminophenols , Catalysis , Drug Design , Enzymes/chemical synthesis , Enzymes/chemistry , Iron , Kinetics , Molecular Mimicry , Monophenol Monooxygenase/chemistry , Oxygen/metabolism , Quinones/chemical synthesis , Structure-Activity Relationship
4.
Biochem Soc Trans ; 32(Pt3): 412-5, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15157149

ABSTRACT

We have expressed two proteins that correspond to the transmembrane and cytoplasmic domains of integrin alphaIIb and beta3 subunits. Characterization of these proteins, dispersed in anionic and zwitterionic micelles, revealed that, rather than interacting with each other, the two proteins associated into homodimers and homotrimers respectively. Moreover, studies using the TOXCAT assay system confirmed that the alphaIIb and beta3 transmembrane domains can self-associate in biological cell membranes. Transmembrane domain-mediated homo-oligomerization provides a plausible structural basis for integrin clustering and could promote integrin activation as well. Indeed, replacing specific residues in the transmembrane helix of either alphaIIb or beta3 with an asparagine residue resulted in a facilitated homo-oligomerization of the mutated transmembrane helix, promoted the formation of integrin clusters on the cell surface and shifted alphaIIbbeta3 to its activated state. Thus these studies support the hypothesis that the transmembrane domains play a vital role in the function and regulation of alphaIIbbeta3.


Subject(s)
Platelet Glycoprotein GPIIb-IIIa Complex/chemistry , Amino Acid Motifs , Animals , CHO Cells , Cell Membrane/metabolism , Cricetinae , Cytoplasm/metabolism , Dimerization , Escherichia coli/metabolism , Gene Expression Regulation , Ligands , Micelles , Protein Binding , Protein Conformation , Protein Structure, Tertiary
6.
Chem Rev ; 101(10): 3025-6, 2001 Oct 10.
Article in English | MEDLINE | ID: mdl-11749399
7.
J Am Chem Soc ; 123(51): 12749-57, 2001 Dec 26.
Article in English | MEDLINE | ID: mdl-11749531

ABSTRACT

De novo design of proteins provides an attractive approach to uncover the essential features required for their functions. Previously, we described the design and crystal structure determination of a di-Zn(II) complex of "due-ferri-1" (DF1), a protein patterned after the diiron-dimanganese class of redox-active proteins [Lombardi, A.; Summa, C.; Geremia, S.; Randaccio, L.; Pavone, V.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6298-6305]. The overall structure of DF1, which contains a carboxylate-bridged dinuclear metal site, agrees well with the intended design. However, access to this dimetal site is blocked by a pair of hydrophobic leucine residues (L13 and L13'), which prevent facile entry of metal ions and small molecules. We have now taken the next step in the eventual construction of a catalytically active metalloenzyme by engineering an active site cavity into DF1 through the replacement of these two leucine residues with smaller residues. The crystal structure of the dimanganous form of L13A-DF1 indeed shows a substrate access channel to the dimetal center. In the crystal structure, water molecules and a ligating dimethyl sulfoxide molecule, which forms a monatomic bridge between the metal ions, occupy the cavity. Furthermore, the diferric form of a derivative of L13A-DF1, DF2, is shown to bind azide, acetate, and small aromatic molecules.


Subject(s)
Metalloproteins/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Ligands , Mixed Function Oxygenases/chemistry , Models, Molecular , Molecular Sequence Data , Protein Conformation , Ribonucleotide Reductases/chemistry
10.
Proc Natl Acad Sci U S A ; 98(22): 12462-7, 2001 Oct 23.
Article in English | MEDLINE | ID: mdl-11606749

ABSTRACT

Integrins are a family of alpha/beta heterodimeric membrane proteins, which mediate cell-cell and cell-matrix interactions. The molecular mechanisms by which integrins are activated and cluster are currently poorly understood. One hypothesis posits that the cytoplasmic tails of the alpha and beta subunits interact strongly with one another in a 1:1 interaction, and that this interaction is modulated in the course of the activation of alphaIIbbeta3 [Hughes, P. E., et al. (1996) J. Biol. Chem. 271, 6571-6574]. To examine the structural basis for this interaction, protein fragments encompassing the transmembrane helix plus cytoplasmic tails of the alpha and beta subunits of alphaIIbbeta3 were expressed and studied in phospholipid micelles at physiological salt concentrations. Analyses of these fragments by analytical ultracentrifugation, NMR, circular dichroism, and electrophoresis indicated that they had very little or no tendency to interact with one another. Instead, they formed homomeric interactions, with the alpha- and beta-fragments forming dimers and trimers, respectively. Thus, these regions of the protein structure may contribute to the clustering of integrins that accompanies cellular adhesion.


Subject(s)
Cytoplasm/chemistry , Membrane Proteins/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex/chemistry , Amino Acid Sequence , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Magnetic Resonance Spectroscopy , Micelles , Molecular Sequence Data , Ultrafiltration
11.
Biochemistry ; 40(32): 9560-9, 2001 Aug 14.
Article in English | MEDLINE | ID: mdl-11583155

ABSTRACT

Understanding how the amino acid sequence of a polypeptide chain specifies a unique, functional three-dimensional structure remains an important goal, especially in the context of the emerging discipline of de novo protein design. Alpha3D is a single chain protein of 73 amino acids resulting from a de novo design effort. Previous solution nuclear magnetic resonance studies of alpha3D confirm that the protein adopts the designed structure of a three-helix bundle. Furthermore, alpha3D has been previously shown to possess all of the major thermodynamic and structural characteristics of natural proteins, though it shares no sequence homology to any protein sequence in the database. In this work, the backbone and side-chain dynamics of alpha3D were investigated using 15N, 13C, and 2H nuclear magnetic resonance relaxation methods with the aim of assessing the character of the internal motions of this native-like protein of de novo design. At the backbone level, both 15N and 13C(alpha) relaxation studies indicate highly restrictive motion on the picosecond to nanosecond time scale in the alpha-helical regions of alpha3D, with increasing mobility at the ends of the alpha-helices and in the two loop regions. This is largely consistent with what is seen in proteins of natural origin. Overall, the view provided by both 2H and 13C methyl relaxation methods suggest that the side chains of alpha3D are more dynamic compared to natural proteins. Regions of relative flexibility bound clusters of rigid methyl-bearing side-chain groups that are interspersed with aromatic and beta-branched amino acids. The time scale of motions associated with methyl-bearing side chains of alpha3D are significantly longer than that seen in natural proteins. These results indicate that the strategies underlying the design of alpha3D have largely, but not completely, captured both the structural and dynamic character of natural proteins.


Subject(s)
Proteins/chemistry , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Engineering , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary
12.
J Mol Biol ; 311(5): 1081-90, 2001 Aug 31.
Article in English | MEDLINE | ID: mdl-11531341

ABSTRACT

Antiparallel helical bundles are found in a wide range of proteins. Often, four-helical bundles form tube-like structures, with binding sites for substrates or cofactors near their centers. For example, a transmembrane four-helical bundle in cytochrome bc(1) binds a pair of porphyrins in an elongated central cavity running down the center of the structure. Antiparallel helical barrels with larger diameters are found in the crystal structures of TolC and DSD, which form antiparallel 12-helical and six-helical bundles, respectively. The backbone geometries of the helical bundles of cytochrome bc(1), TolC, and DSD are well described using a simple D(n)-symmetrical model with only eight adjustable parameters. This parameterization provides an excellent starting point for construction of minimal models of these proteins as well as the de novo design of proteins with novel functions.


Subject(s)
Models, Molecular , Proteins/chemistry , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Binding Sites , Coenzymes/metabolism , Dimerization , Electron Transport Complex III/chemistry , Electron Transport Complex III/metabolism , Escherichia coli Proteins , Ions/metabolism , Ligands , Membrane Transport Proteins , Protein Structure, Secondary , Protein Structure, Tertiary , Proteins/metabolism
13.
J Am Chem Soc ; 123(38): 9235-8, 2001 Sep 26.
Article in English | MEDLINE | ID: mdl-11562202

ABSTRACT

The helix-coil transition of a synthetic alpha-helical peptide (the D-Arg peptide), Ac-YGG(KAAAA)(3)-CO-D-Arg-CONH(2), was studied by static far-UV circular dichroism (CD) and time-resolved infrared spectroscopy coupled with the laser-induced temperature-jump technique for rapid relaxation initiation. Equilibrium thermal unfolding measurements of the D-Arg peptide monitored by CD spectroscopy reveal an apparent two-state helix-coil transition, with a thermal melting temperature around 10 degrees C. Time-resolved infrared (IR) measurements following a laser-induced temperature jump, however, reveal biphasic (or multiphasic) relaxation kinetics. The fast phase rises within the 20 ns response time of the detection system. The slow phase has a decay lifetime of approximately 140 ns at 300 K and exhibits monotonic temperature dependence with an apparent activation energy around 15.5 kcal/mol.


Subject(s)
Alanine/chemistry , Peptides/chemistry , Protein Folding , Arginine/chemistry , Circular Dichroism , Kinetics , Protein Structure, Secondary , Spectroscopy, Fourier Transform Infrared , Temperature
14.
Biochem Soc Trans ; 29(Pt 4): 559-64, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11498028

ABSTRACT

Our current level of understanding of membrane-protein folding is primitive, but it is beginning to advance. Previously [Choma, Gratkowski, Lear and DeGrado (2000) Nat. Struct. Biol. 7, 161-166], we described studies of the association in detergent micelles of short, simple-sequence hydrophobic peptides modified from the sequence of the water-soluble, homodimeric coiled-coil GCN4-P1 peptide using the principle that the interiors of membrane proteins are similar to those of water-soluble proteins. Here, we discuss more quantitative aspects of the association equilibrium and compare the free energies of association of a number of mutant peptides designed to explore specific features responsible for the association.


Subject(s)
DNA-Binding Proteins/biosynthesis , Fungal Proteins/biosynthesis , Membrane Proteins/biosynthesis , Peptides/metabolism , Protein Kinases/biosynthesis , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Amino Acid Substitution , DNA-Binding Proteins/chemistry , Dimerization , Drug Design , Fungal Proteins/chemical synthesis , Fungal Proteins/chemistry , Kinetics , Membrane Proteins/chemical synthesis , Membrane Proteins/chemistry , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptides/chemistry , Protein Folding , Protein Kinases/chemical synthesis , Protein Kinases/chemistry , Protein Structure, Secondary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry
15.
J Am Chem Soc ; 123(31): 7553-9, 2001 Aug 08.
Article in English | MEDLINE | ID: mdl-11480975

ABSTRACT

beta-Peptides are a class of polyamides that have been demonstrated to adopt a variety of helical conformations. Recently, a series of amphiphilic L(+2) helical beta-peptides were designed, which were intended to mimic the overall physicochemical properties of a class of membrane-active antimicrobial peptides, including magainin and cecropin. Although these peptides showed potent antimicrobial activity, they also showed significant activity against human erythrocytes. Operating under the assumption that their lack of specificity arose from excessive hydrophobicity, two additional beta-peptides H-(beta(3)-HAla-beta(3)-HLys-beta(3)-HVal)(n)-NH(2) (n = 4, 5) were designed and synthesized. Both have high antimicrobial activities, but very low hemolytic potencies. The peptides bind in an L(+2) conformation to phospholipid vesicles, inducing leakage of entrapped small molecules. The peptides have a low affinity for membranes consisting of neutral phosphatidylcholine lipids, but bind avidly to vesicles containing 10 mol % of acidic phosphatidylserine lipids. Differences in vesicle leakage kinetics for the two peptides suggest that chain length could affect their mechanisms of disrupting cell membranes. Thus, insights gained from the study of variants of natural alpha-peptides have provided a useful guide for the design of nonnatural antimicrobial beta-peptides.


Subject(s)
Anti-Infective Agents/chemical synthesis , Antimicrobial Cationic Peptides/chemical synthesis , Drug Design , Antimicrobial Cationic Peptides/chemistry , Circular Dichroism , Liposomes , Molecular Structure , Protein Binding , Protein Folding
17.
Protein Sci ; 10(5): 958-69, 2001 May.
Article in English | MEDLINE | ID: mdl-11316876

ABSTRACT

DF1 is a small, idealized model for carboxylate-bridged diiron proteins. This protein was designed to form a dimeric four-helix bundle with a dimetal ion-binding site near the center of the structure, and its crystal structure has confirmed that it adopts the intended conformation. However, the protein showed limited solubility in aqueous buffer, and access to its active site was blocked by two hydrophobic side chains. The sequence of DF1 has now been modified to provide a very soluble protein (DF2) that binds metal ions in a rapid and reversible manner. Furthermore, the DF2 protein shows significant ferroxidase activity, suggesting that its dimetal center is accessible to oxygen. The affinity of DF2 for various first-row divalent cations deviates from the Irving-Willliams series, suggesting that its structure imparts significant geometric preferences on the metal ion-binding site. Furthermore, in the absence of metal ions, the protein folds into a dimer with concomitant binding of two protons. The uptake of two protons is expected if the structure of the apo-protein is similar to that of the crystal structure of dizinc DF1. Thus, this result suggests that the active site of DF2 is retained in the absence of metal ions.


Subject(s)
Cations, Divalent/pharmacology , Nonheme Iron Proteins/chemistry , Nonheme Iron Proteins/metabolism , Amino Acid Sequence , Amino Acid Substitution/genetics , Binding Sites , Cations, Divalent/metabolism , Dimerization , Guanidine/pharmacology , Hydrogen-Ion Concentration , Iron/metabolism , Models, Molecular , Molecular Sequence Data , Nonheme Iron Proteins/chemical synthesis , Nonheme Iron Proteins/genetics , Oxidation-Reduction/drug effects , Oxygen/metabolism , Protein Binding , Protein Denaturation/drug effects , Protein Folding , Protein Structure, Quaternary/drug effects , Protein Structure, Secondary/drug effects , Protein Subunits , Protons , Static Electricity , Thermodynamics , Ultracentrifugation
18.
Biochemistry ; 40(9): 2790-6, 2001 Mar 06.
Article in English | MEDLINE | ID: mdl-11258889

ABSTRACT

Substitution of leucine residues by 5,5,5-trifluoroleucine at the d-positions of the leucine zipper peptide GCN4-p1d increases the thermal stability of the coiled-coil structure. The midpoint thermal unfolding temperature of the fluorinated peptide is elevated by 13 degrees C at 30 microM peptide concentration. The modified peptide is more resistant to chaotropic denaturants, and the free energy of folding of the fluorinated peptide is 0.5-1.2 kcal/mol larger than that of the hydrogenated form. A similarly fluorinated form of the DNA-binding peptide GCN4-bZip binds to target DNA sequences with affinity and specificity identical to those of the hydrogenated form, while demonstrating enhanced thermal stability. Molecular dynamics simulation on the fluorinated GCN4-p1d peptide using the Surface Generalized Born implicit solvation model revealed that the coiled-coil binding energy is 55% more favorable upon fluorination. These results suggest that fluorination of hydrophobic substructures in peptides and proteins may provide new means of increasing protein stability, enhancing protein assembly, and strengthening receptor-ligand interactions.


Subject(s)
Fungal Proteins/chemistry , Fungal Proteins/metabolism , Leucine Zippers , Leucine/analogs & derivatives , Leucine/chemistry , Peptides/chemistry , Peptides/metabolism , Protein Kinases/chemistry , Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Circular Dichroism , DNA/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Dimerization , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Peptides/chemical synthesis , Protein Structure, Tertiary , Static Electricity , Thermodynamics
19.
Proc Natl Acad Sci U S A ; 98(3): 880-5, 2001 Jan 30.
Article in English | MEDLINE | ID: mdl-11158564

ABSTRACT

The forces stabilizing the three-dimensional structures of membrane proteins are currently not well understood. Previously, it was shown that a single Asn side chain in a transmembrane segment can mediate the dimerization and trimerization of a variety of hydrophobic helices. Here, we examine the tendencies of a representative set of amino acids (Asn, Gln, Asp, Glu, Lys, Ala, Val, Leu, Ser, Thr) to direct the oligomerization of a model transmembrane helix. The model peptide is entirely hydrophobic throughout a 20-residue segment and contains a single central site for the introduction of various amino acid "guests." Analytical ultracentrifugation and gel electrophoresis were used to determine the stoichiometry and free energy of association of the entire set of peptides within micelles. Variants with two polar atoms at the guest site-Asn, Gln, Asp, and Glu-formed stable trimers, whereas residues with one or fewer polar atoms showed a much weaker tendency to associate. The data are examined in light of the frequencies of occurrence of various amino acid side chains in membrane proteins and provide insight into the role of polar interactions in directing transmembrane helix association. These data also suggest an approach to the design of variants of natural single-span transmembrane proteins with various potentials to associate in the bilayer.


Subject(s)
Membrane Proteins/chemistry , Peptides/chemistry , Protein Structure, Secondary , Amino Acid Sequence , Amino Acids , Detergents , Electrophoresis, Polyacrylamide Gel , Models, Chemical , Molecular Sequence Data , Peptides/chemical synthesis , Structure-Activity Relationship , Ultracentrifugation
20.
Proc Natl Acad Sci U S A ; 98(4): 1404-9, 2001 Feb 13.
Article in English | MEDLINE | ID: mdl-11171963

ABSTRACT

Three-dimensional (3D) domain-swapped proteins are intermolecularly folded analogs of monomeric proteins; both are stabilized by the identical interactions, but the individual domains interact intramolecularly in monomeric proteins, whereas they form intermolecular interactions in 3D domain-swapped structures. The structures and conditions of formation of several domain-swapped dimers and trimers are known, but the formation of higher order 3D domain-swapped oligomers has been less thoroughly studied. Here we contrast the structural consequences of domain swapping from two designed three-helix bundles: one with an up-down-up topology, and the other with an up-down-down topology. The up-down-up topology gives rise to a domain-swapped dimer whose structure has been determined to 1.5 A resolution by x-ray crystallography. In contrast, the domain-swapped protein with an up-down-down topology forms fibrils as shown by electron microscopy and dynamic light scattering. This demonstrates that design principles can predict the oligomeric state of 3D domain-swapped molecules, which should aid in the design of domain-swapped proteins and biomaterials.


Subject(s)
Oligopeptides/chemistry , Dimerization , Protein Folding , Protein Structure, Tertiary
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