Protein minimization: downsizing through mutation.
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Kinetics↗
Biomedical subjects
W F DeGrado
Publications and source records attributed to W F DeGrado.
Controlling topology and native-like behavior of de novo-designed peptides: design and characterization of antiparallel four-stranded coiled coils.
The de novo design of peptides and proteins has emerged as an attractive approach for investigating protein structure and function. Here, the design, synthesis, and characterization of a new series of alpha-helical peptides intended to form antiparallel four-stranded coiled coils is described. Computer models were generated without the use of extant protein structures and were used to refine the sequence. The peptides are of the general formula Ncap-(XaZbZcLdZeZfZg)3-Ccap, where X is either Ala, Val, Thr, or Leu, and Ncap and Ccap are sequences designed to satisfy the helices unpaired amide nitrogens and carbonyl oxygens, respectively. The hydrophobic residues (at positions a and d) were chosen so that geometric packing of large and small hydrophobes would favor an antiparallel arrangement. Special attention was also given to residues at the helix--helix interfaces. These residues were chosen to balance potential attractive and repulsive electrostatic forces so that the desired topology was favored while other possible folds were destabilized. Two of the four peptides associate under neutral conditions into the desired tetramers. One of the complexes (a = Val) behaves like a native-like protein as judged by NMR, thermodynamics, and apolar dye (ANS) binding. The other tetrameric complex (a = Leu) exhibits broader NMR resonances, diminished values of delta H and delta Cp, and tight binding of the hydrophobic dye ANS, similar to early designed proteins. These results reinforce the importance of optimizing van der Waals packing interactions in protein design but demonstrate that hydrophobic packing must be balanced with hydrogen-bonding and electrostatic interactions to produce novel native-like proteins.
Amino Acid Sequence↗
Oral antiplatelet, antithrombotic efficacy of DMP 728, a novel platelet GPIIb/IIIa antagonist.
BACKGROUND: Currently used antiplatelet drugs, including aspirin and ticlopidine, are effective against certain but not all of the many endogenous platelet activators. Because of their limited efficacy, a significant number of serious thromboembolic complications still occur, highlighting the need for a more effective therapy. DMP 728 has been characterized as a potent and specific platelet glycoprotein IIb/IIIa complex (GPIIb/IIIa) antagonist. The goals of the present study were to determine the oral antiplatelet and antithrombotic efficacies of DMP 728 in various arterial thrombosis models in dogs. METHODS AND RESULTS: In conscious and anesthetized mongrel dogs, DMP 728 at 0.02 to 1.0 mg/kg PO in gelatin capsules produced dose-dependent antiplatelet effects in inhibiting ex vivo platelet aggregation induced by ADP and prolonging template bleeding time. DMP 728 effects on bleeding time prolongation could be reversed more rapidly than those on platelet aggregation inhibition. A maximal antiplatelet effect for DMP 728 was demonstrated at 1.0 mg/kg PO. DMP 728 demonstrated dose-dependent oral antiplatelet effects with an absolute oral bioavailability of 8% to 12% in dogs. Additionally, the antithrombotic efficacy of DMP 728 was examined after intravenous and oral administration at different doses in various models of arterial thrombosis. In the coronary artery Folts' model in dogs, DMP 728 demonstrated maximal antithrombotic efficacy at 0.01 mg/kg IV and < 0.6 mg/kg PO. Additionally, DMP 728 at 0.1 and 1.0 mg/kg IV or PO demonstrated 60% to 100% prevention of primary thrombosis (P < .01) in an electrolytically induced carotid artery thrombosis model in dogs. CONCLUSIONS: These data suggest that DMP 728, a low-molecular-weight GPIIb/IIIa receptor antagonist, may have therapeutic potential as an oral antithrombotic agent in coronary and carotid artery thromboembolic disorders.
Adenosine Diphosphate↗
The role of helix formation in the folding of a fully alpha-helical coiled coil.
To determine when secondary structure forms as two chains coalesce to form an alpha-helical dimer, the folding rates of variants of the coiled coil region of GCN4 were compared. Residues at non-perturbing positions along the exterior length of the helices were substituted one at a time with alanine and glycine to vary helix propensity and therefore dimer stability. For all variants, the bimolecular folding rate remains largely unchanged; the unfolding rate changes to largely account for the change in stability. Thus, contrary to most folding models, widespread helix is not yet formed at the rate-limiting step in the folding pathway. The high-energy transition state is a collapsed form that contains little if any secondary structure, as suggested for the globular protein cytochrome c (Sosnick et al., Proteins 24: 413-426, 1996).
Amino Acid Sequence↗
De novo design of heterotrimeric coiled coils.
The three-helix bundle is a common structural motif among natural proteins. It has been observed in numerous important proteins, such as fibrinogen, laminin, spectrin, dystrofin, hemagglutinin, and mannose binding proteins. The three-helix bundle is a simple structure in which three alpha-helices pack against each other, with a slight left-handed twist. Because of its simplicity relative to other structural motifs, the three-helix bundle can be conveniently used both to clarify the forces responsible for the protein folding and stability, and for the design of novel proteins. In this paper we describe the design, synthesis, and characterization of three peptides that self-assemble into antiparallel, heterotrimeric coiled coils. The experimental results, obtained from CD spectroscopy and ultracentrifugation equilibrium sedimentation, indicate that the mixture of the three peptides preferentially forms heterotrimers; moreover, these aggregates represent attractive systems for combinatorial design of libraries of pseudo C3 symmetric ligands or binding sites.
Amino Acid Sequence↗
Crystallization of a designed peptide from a molten globule ensemble.
BACKGROUND: The design of amino acid sequences that adopt a desired three-dimensional fold has been of keen interest over the past decade. However, the design of proteins that adopt unique conformations is still a considerable problem. Until very recently, all of the designed proteins that have been extensively characterized possess the hallmarks of the molten globular state. Molten globular intermediates have been observed in both equilibrium and kinetic protein folding/stability studies, and understanding the forces that determine compact non-native states is critical for a comprehensive understanding of proteins. This paper describes the solution and early solid state characterization of peptides that form molten globular ensembles. RESULTS & CONCLUSIONS: Crystals diffracting to 3.5 A resolution have been grown of a 16-residue peptide (alpha 1A) designed to form a tetramer of alpha-helices. In addition, a closely related peptide, alpha 1, has previously been shown to yield crystals that diffract to 1.2 A resolution. The solution properties of these two peptides were examined to determine whether their well defined crystalline conformations were retained in solution. On the basis of an examination of their NMR spectra, sedimentation equilibria, thermal unfolding, and ANS binding, it is concluded that the peptides form alpha-helical aggregates with properties similar to those of the molten globule state. Thus, for these peptides, the process of crystallization bears many similarities to models of protein folding. Upon dissolution, the peptides rapidly assume compact molten globular states similar to the molten globule like intermediates that are formed at short times after refolding is initiated. Following a rate-determining nucleation step, the peptides crystallize into a single or a small number of conformations in a process that mimics the formation of native structure in proteins.
Amino Acid Sequence↗
In vitro evolution of thermodynamically stable turns.
To determine the role of primary structure in specifying turns, random sequences (guests) were substituted for the native turn sequences in a series of proteins (hosts) of differing thermodynamic stabilities.The fraction of inserts that result in active proteins is measured as a function of the stability of the host and temperature. With a highly stable host, more than half of the inserts give functional proteins. However, a smaller fraction of sequences supports folding as the stability of the host decreases, and the temperature increases. The sequences of many of the selected inserts resemble the wild-type turn, and those that diverge match other established turn preferences. Thermodynamic measurements show that turn sequences selected under stringent conditions result in the most stable proteins. Thus, beta-turns appear to be under evolutionary pressure favouring thermodynamically stable structures.
Amino Acid Sequence↗
Expression of de novo designed alpha-helical bundles.
The successful design of proteins requires careful consideration of the multiplicity of forces that stabilize their three-dimensional structures including hydrophobic interactions, hydrogen-bonding, electrostatics and weakly polar interactions. Early attempts to design proteins relied too heavily on hydrophobic interactions to provide stability, resulting in structures with dynamic properties. Addition of more specific interactions to these initial designs gives rise to proteins with more native-like properties. This manuscript describes the design of native-like three- and four-helix bundles, and their cloning and expression of these proteins.
Amino Acid Sequence↗
Protein design: a hierarchic approach.
The de novo design of peptides and proteins has recently emerged as an approach for investigating protein structure and function. Designed, helical peptides provide model systems for dissecting and quantifying the multiple interactions that stabilize secondary structure formation. De novo design is also useful for exploring the features that specify the stoichiometry and stability of alpha-helical coiled coils and for defining the requirements for folding into structures that resemble native, functional proteins. The design process often occurs in a series of discrete steps. Such steps reflect the hierarchy of forces required for stabilizing tertiary structures, beginning with hydrophobic forces and adding more specific interactions as required to achieve a unique, functional protein.
Amino Acid Sequence↗
Thermodynamic genetics of the folding of the B1 immunoglobulin-binding domain from streptococcal protein G.
A method has been developed to select proteins that are thermodynamically destabilized yet still folded and functional. The DNA encoding the B1 IgG-binding domain from Group G Streptococcus (Strp G) has been fused to gene III of bacteriophage M13. The resulting fusion protein is displayed on the surface of the phage thus enabling the phage to bind to IgG molecules. In addition, these phage exhibit a small plaque phenotype that is reversed by mutations that destabilize the Strp G domain. By selecting phage with large plaque morphology that retain their IgG-binding function, it is possible to identify mutants that are folded but destabilized compared with wild-type Strp G. Such mutants can be divided into three general categories: 1) those that disrupt packing of hydrophobic side chains in the protein interior; 2) those that destabilize secondary structure; and 3) those that alter specific hydrogen bonds involving amino acid side chains. A number of the mutants have been physically characterized by circular dichroism and nuclear magnetic resonance and have been shown to have structures similar to wild-type Strp G but stabilities that were decreased by 2-5 kcal/mol.
Amino Acid Sequence↗
Native-like and structurally characterized designed alpha-helical bundles.
A number of coiled coils and alpha-helical bundles have recently been designed, and many have now been structurally characterized by X-ray crystallography. Others have not been as well characterized structurally but exhibit native-like properties in aqueous solution. Both areas of investigation have contributed greatly to our understanding of the nature of specificity in this class of molecules.
Amino Acid Sequence↗
A helical-dipole model describes the single-channel current rectification of an uncharged peptide ion channel.
We are designing simple peptide ion channels as model systems for the study of the physical principles controlling conduction through ion-channel proteins. Here we report on an uncharged peptide, Ac-(Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2, designed to form an aggregate of parallel, amphiphilic, membrane-spanning alpha-helices around a central water-filled pore. This peptide in planar lipid bilayers forms ion channels that show single-channel current rectification in symmetric 1 M KCl. The current at a given holding membrane potential is larger than the current measured through the same channel when the potential is reversed. Based on our hypothesized gating mechanism, the larger currents flow from the peptide carboxyl terminus toward the amino terminus. We present an ionic electrodiffusion model based on the helical-dipole potential and the dielectric interfacial polarization energy, which with reasonable values for dipole magnitude and dielectric constants, accurately replicates the current-voltage data.
Amino Acid Sequence↗
Design and synthesis of multi-haem proteins.
A water-soluble, 62-residue, di-alpha-helical peptide has been synthesized which accommodates two bis-histidyl haem groups. The peptide assembles into a four-helix dimer with 2-fold symmetry and four parallel haems that closely resemble native haems in their spectral and electrochemical properties, including haem-haem redox interaction. This protein is an essential intermediate in the synthesis of molecular 'maquettes', a novel class of simplified versions of the metalloproteins involved in redox catalysis and in energy conversion in respiratory and photosynthetic electron transfer.
Amino Acid Sequence↗
Identification of recognition sequences of adhesion molecules using phage display technology.
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Amino Acid Sequence↗
Design and synthesis of simplified energy-converting proteins.
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Amino Acid Sequence↗
Multiple oligomeric states regulate the DNA binding of helix-loop-helix peptides.
To study the protein-protein interactions that allow Id, a negative regulator of cell differentiation, to inhibit the DNA-binding activities of MyoD and E47, we have synthesized peptides corresponding to the helix-loop-helix domains of MyoD, E47, and Id. We show that Id preferentially inhibits the sequence-specific DNA-binding activity of MyoD, a muscle-specific protein, as compared to E47, a more ubiquitous protein. The Id helix-loop-helix domain itself forms stable tetramers, and its inhibitory activity arises from the formation of a heterotetrameric structure with MyoD. The formation of this higher order complex provides a general mechanism by which inhibitory proteins can generate sufficient interaction free energy to overcome the large DNA-binding free energy of dimeric DNA-binding proteins.
Amino Acid Sequence↗
Peptide engineering. Catalytic molten globules.
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Amino Acid Sequence↗
Metal ion-dependent modulation of the dynamics of a designed protein.
The peptide alpha 4 is a designed four-helix bundle that contains a highly simplified hydrophobic core composed exclusively of leucine residues; its tertiary structure is therefore largely dictated by hydrophobic forces. This small protein adopts a structure with properties intermediate between those of the native and molten globule states of proteins: it is compact, globular, and has very stable helices, but its apolar side chains are mobile and not as well packed as in many natural proteins. To induce a more native-like state, two Zn(2+)-binding sites were introduced into the protein, thereby replacing some of the non-specific hydrophobic interactions with more geometrically restrictive metal-ligand interactions. In the metal-bound state, this protein has properties that approach those of native proteins. Thus, hydrophobic interactions alone are sufficient to drive polypeptide chain folding nearly to completion, but specific interactions are required for a unique structure.
Amino Acid Sequence↗