Design and Characterization of an Intramolecular Antiparallel Coiled Coil Peptide

Myszka, David G.; Chaiken, Irwin M.

doi:10.1021/bi00175a003

Cited by 78 publications

(49 citation statements)

References 52 publications

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“…Serine was chosen because of its relatively small size, polar character, and tolerance for inclusion into ␣-helices (21,22). The intended effect of the sequential substitutions is to gradually remove the potential hydrophobic-binding site while retaining intact helices and the overall rod-like structure of the tentacle.…”

Section: Cepfd6mentioning

confidence: 99%

Molecular clamp mechanism of substrate binding by hydrophobic coiled-coil residues of the archaeal chaperone prefoldin

Lundin

Stirling

Gόmez-Reino

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Prefoldin (PFD) is a jellyfish-shaped molecular chaperone that has been proposed to play a general role in de novo protein folding in archaea and is known to assist the biogenesis of actins, tubulins, and potentially other proteins in eukaryotes. Using point mutants, chimeras, and intradomain swap variants, we show that the six coiledcoil tentacles of archaeal PFD act in concert to bind and stabilize nonnative proteins near the opening of the cavity they form. Importantly, the interaction between chaperone and substrate depends on the mostly buried interhelical hydrophobic residues of the coiled coils. We also show by electron microscopy that the tentacles can undergo an en bloc movement to accommodate an unfolded substrate. Our data reveal how archael PFD uses its unique architecture and intrinsic coiled-coil properties to interact with nonnative polypeptides.C oiled coils consist of two or more parallel or antiparallel amphipathic ␣-helices that twist around one another to form supercoils (1). The primary sequences of the helices display a heptad repeat (abcdefg), where apolar residues are found preferentially in the first (a) and fourth (d) positions. Although the knobs-into-holes packing of the hydrophobic residues is the predominant stabilizing force for a coiled coil, inter-and intrahelical ionic interactions can act to further stabilize or destabilize its supersecondary structure (1). Coiled coils are found in several molecular chaperones, a diverse family of proteins whose collective cellular role is to ensure the quality control (e.g., folding, assembly, and transport) of nonnative proteins (2, 3). Archaeal prefoldin (PFD) is a chaperone that contains six canonical antiparallel coiled coils whose N-and C-terminal helices project outward from a double ␤-barrel oligomerization domain; the overall shape of the hexameric protein complex, assembled from two PFD␣ and four PFD␤ subunits (␣ 2 ␤ 4 ), resembles a jellyfish with six tentacles (4). In solution, its tentacles are likely to be fully solvated and independently mobile (4). A lower-resolution electron microscope image of recombinant human PFD, which consists of six different proteins (two ␣ class and four ␤ class subunits), reveals that it possesses the same overall structure (5).Like other chaperones, archaeal PFD can selectively interact with and stabilize nonnative (unfolded) polypeptides that expose hydrophobic surfaces in vitro, helping to prevent their aggregation (4, 6, 7). Preliminary studies have shown that deletion of the distal coiled-coil regions in either the ␣ or ␤ subunit abrogates chaperone activity in vitro, implying that PFD grasps its substrates in a multivalent manner (4). Similarly, the eukaryotic PFD-actin complex recently visualized by EM shows that nonnative actin, one of its substrates, makes multiple contacts with the distal regions of the tentacles (5).In the crowded cellular environment, eukaryotic PFD is likely to transiently stabilize ribosome-bound nascent polypeptides (8) before shuttling them to a chaperonin (an ATP-depe...

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Section: Cepfd6mentioning

confidence: 99%

Molecular clamp mechanism of substrate binding by hydrophobic coiled-coil residues of the archaeal chaperone prefoldin

Lundin

Stirling

Gόmez-Reino

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…The R-helices composing the coiled-coil structure are defined by a heptad repeat (denoted abcdefg) in which hydrophobic residues occupy the positions a and d and pack in a characteristic "knobs-intoholes" manner (6), forming the hydrophobic core ( Figure 1). Many studies (1,(7)(8)(9)(10)(11)(12) emphasized the role of charged residues (typically in the e and g positions) in controlling the specificity of oligomerization and opened up the way to the de novo design of heterodimeric coiled-coils (13)(14)(15)(16)(17). Their relatively small size, in addition to their well-defined structure, make coiled-coils a very attractive system for protein engineering and biotechnological applications (5,18) such as the construction of miniaturized antibodies, the control of signaling protein domain oligomerization, and the specific targeting of GFP to cytoskeletal proteins (see ref 4 for review).…”

mentioning

confidence: 99%

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

et al. 2003

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We have de novo designed a heterodimeric coiled-coil formed by two peptides as a capture/ delivery system that can be used in applications such as affinity tag purification, immobilization in biosensors, etc. The two strands are designated as K coil (KVSALKE heptad sequence) and E coil (EVSALEK heptad sequence), where positively charged or negatively charged residues occupy positions e and g of the heptad repeat. In this study, for each E coil or K coil, three peptides were synthesized with lengths varying from three to five heptads. The effect of the chain length of each partner upon the kinetic and thermodynamic constants of interaction were determined using a surface plasmon resonance-based biosensor. Global fitting of the interactions revealed that the E5 coil interacted with the K5 coil according to a simple binding model. All the other interactions involving shorter coils were better described by a more complex kinetic model involving a rate-limiting reorganization of the coiled-coil structure. The affinities of these de novo designed coiled-coil interactions were found to range from 60 pM (E5/K5) to 30 µM (E3/K3). From these K d values, we were able to determine the free energy contribution of each heptad, depending on its relative position within the coiled-coils. We found that the free energy contribution of a heptad occupying a central position was 3-fold higher than that of a heptad at either end of the coiled-coil. The wide range of stabilities and affinities for the E/K coil system provides considerable flexibility for protein engineering and biotechnological applications.The R-helical coiled-coil is an oligomerization domain that is naturally present in a wide variety of proteins such as transcription factors, cytoskeletal proteins, motor proteins, and viral fusion proteins (1-3). The structural properties of coiled-coils have been extensively characterized from numerous high-resolution crystal and NMR structures (3-5). The formation of coiled-coils requires the wrapping of two or more amphipathic R-helices around each other in a lefthanded supercoil fashion; the helices may be aligned in either a parallel or antiparallel manner. The R-helices composing the coiled-coil structure are defined by a heptad repeat (denoted abcdefg) in which hydrophobic residues occupy the positions a and d and pack in a characteristic "knobs-intoholes" manner (6), forming the hydrophobic core ( Figure 1). Many studies (1,(7)(8)(9)(10)(11)(12) emphasized the role of charged residues (typically in the e and g positions) in controlling the specificity of oligomerization and opened up the way to the de novo design of heterodimeric coiled-coils (13-17). Their relatively small size, in addition to their well-defined structure, make coiled-coils a very attractive system for protein engineering and biotechnological applications (5, 18) such as the construction of miniaturized antibodies, the control of signaling protein domain oligomerization, and the specific targeting of GFP to cytoskeletal proteins (see ref 4 for ...

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“…An amphiphilic peptide containing minimalist helical regions (Lys-Lys-Leu-Leu) and an Asn-Pro-Gly turn region was developed and used to study conformational changes induced by salt and pH (Goto & Aimoto, 1991). A 56-residue antiparallel coiled-coil peptide consisting of minimalist helices connected by a turn region (-Gly-Arg-GlyAsp-Met-Pro-) was characterized by CD and was shown to easily form a disulfide bond between cystines at the amino and carboxy termini (Myszka & Chaiken, 1994). A 35-residue helixloop-helix peptide, ALIN, was designed and characterized by CD and NMR (Kuroda et al,, 1994).…”

Section: Rationale Goals Definition Of Success and Approachmentioning

confidence: 99%

“…However, in this structure the arrangement of the hydrophobic side chains definitely favored dimerization to a four-helix bundle, thus a good example of a narrow antiparallel interface was not available. In work published after the a t a design was completed, peptides with linked antiparallel helices were produced (Monera et al, 1993;Kuroda et al, 1994;Myszka & Chaiken, 1994). After completion of the a t a design, it was noticed that seryl tRNA synthetase contains an antiparallel helical arm (Cusak et al, 1990).…”

Section: Designmentioning

confidence: 99%

Strategies and rationales for the de novo design of a helical hairpin peptide

Fezoui

Weaver²,

Osterhout³

1995

Protein Science

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The de novo design of a t a , a helical hairpin peptide, is described. a t a (u-helix/turn/a-helix) was developed to provide a model system for protein folding at the level of secondary structure association and stabilization. According t o the prevailing models of protein folding, the second step in the folding process is the association and stabilization of secondary structural elements or microdomains. A brief description of the design, along with CD and NMR evidence confirming the conformation of the peptide in solution, has been published (Fezoui Y, Weaver DL, Osterhout JJ, 1994, Proc Natl Acad Sci USA 91:3675-3679). The present work includes a full description of the design process, including the trade-offs that were made during the development of the peptide, a discussion of recent experimental results that were not available at the time of the original design, indications of areas where, in retrospect, the design might have been done differently, and a discussion of how the present work fits into the field of de novo protein design.Keywords: diffusion-collision model; molten globule; NMR; peptide; secondary structure; tertiary structure a t a is a de novo designed 38-amino acid peptide that has been shown by CD and two-dimensional NMR to exhibit in solution the designed aspects of secondary and tertiary structure (Fezoui et al., 1994). The purpose of the present paper is to present a full description of the process that led to the sequence of the peptide. Although the paper cited above contains a brief description of the design, space limitations precluded a complete discussion of the design process. In addition, the original peptide design was completed in the fall of 1990. Since then, there has been considerable progress made in the understanding of peptide conformation and the intervening work would have a bearing on any subsequent design.The goal of the design was to produce a peptide containing two interacting helices connected by a turn region. Such a peptide was desired as an experimental model of secondary structure association that could be used to investigate the early stages of protein folding as envisioned in the diffusion-collision model (Karplus & Weaver, 1976 protein folding are transiently folded elements of secondary structure (microdomains) that associate by diffusion and collision and become mutually stabilizing during the second stage of folding (Karplus & Weaver, 1976.Because the helix-forming properties of isolated monomeric helices had received considerable attention (reviewed by Scholtz & Baldwin, 1992), it seemed reasonable to extend the range of inquiry into the mechanisms of protein folding by studying connected helices. Connected and interacting helices might be expected to be stabilized by both short-range and tertiary interactions. The relative contributions to helix stability among the various short-range and tertiary interactions could be investigated with a helical hairpin peptide. Rationale, goals, definition of success, and approachThe first consideration in un...

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Design and Characterization of an Intramolecular Antiparallel Coiled Coil Peptide

Cited by 78 publications

References 52 publications

Molecular clamp mechanism of substrate binding by hydrophobic coiled-coil residues of the archaeal chaperone prefoldin

Molecular clamp mechanism of substrate binding by hydrophobic coiled-coil residues of the archaeal chaperone prefoldin

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

Strategies and rationales for the de novo design of a helical hairpin peptide

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