α‐Helical coiled coils and bundles: How to design an α‐helical protein

Cohen, Carolyn; Parry, David

doi:10.1002/prot.340070102

Cited by 733 publications

(558 citation statements)

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“…In contrast, the shorter Lac 21 peptide eluted as an asymmetric peak Note that the helix is capped with a proline at the amino-terminus and a glycine at the carboxy-terminus; amino acids that are strong helix-breaking residues and often found at the ends of helices (Richardson & Richardson, 1988). Hydrophobic residues are revealed to be exclusively in a and d positions and are dominated by leucine, a residue commonly found in helical bundle proteins (Cohen & Parry, 1990). We subjected all three peptides to ultracentrifugation at three speeds and the data with their corresponding curve fits are shown in Figure 3B, C, and D. Both the Lac 28 and Lac 35 SE data are fitted using a single species analysis (Fig.…”

Section: Resultsmentioning

confidence: 95%

Characterization of a new four‐chain coiled‐coil: Influence of chain length on stability

et al. 1995

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Limited information is available on inherent stabilities of four-chain coiled-coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four-chain coiled-coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four-chain coiled-coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain a-helical chains 21, 28, and 35 amino acids in length are 3.1 x 6.7 X and 1.0 x M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 pM, 51 nM, and 280 f M , respectively. Finally, we discuss the rules governing coiledcoil formation in light of the work presented here.

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Section: Resultsmentioning

confidence: 95%

Characterization of a new four‐chain coiled‐coil: Influence of chain length on stability

et al. 1995

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“…Examination of amino acid sequences present in a-helical coiled coils and a-helical bundles reveals the common presence of segments containing heptad repeats (abcdefg) with hydrophobic residues at positions a and d. There is no simple rule for assessing the significance of short heptad runs in a sequence. However, according to current structural knowledge, Cohen and Parry (1990) claim that runs of two to three heptads, which have at least 80% hydrophobic occupancy of the a and d positions, have high probability for helix formation. Such a-helices are amphiphatic in nature and are driven together in hydrophilic environments, forming hydrophobic interfaces in a-helical coiled coils or a-helical bundles.…”

Section: Discussionmentioning

confidence: 99%

Subunit interaction in extracellular superoxide dismutase: Effects of mutations in the N‐terminal domain

Stenlund¹,

Andersson²,

Tibell³

1997

Protein Science

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Human extracellular superoxide dismutase (hEC-SOD) is a secreted tetrameric protein involved in protection against oxygen free radicals. Because EC-SOD is too large a protein for structural determination by multidimensional NMR, and attempts to crystallize the protein for X-ray structural determination have failed, the three-dimensional structure of hEC-SOD is unknown. This means that alternative strategies for structural studies are needed. The N-terminal domain of EC-SOD has already been studied using the fusion protein FusNN, comprised of the 49 N-terminal amino acids from hEC-SOD fused to human carbonic anhydrase (HCAII). The N-terminal domain in this fusion protein forms a welldefined three-dimensional structure, which probably contains a-helical elements and is responsible for the tetramerization of the protein.In this work, we have extended the studies, using site-directed mutagenesis in combination with size-exclusion chromatography, CD, and fluorescence spectroscopy, to investigate the nature of the tetrameric interaction. Our results show that the hydrophobic side of a predicted amphiphatic a-helix (formed by residues 14-32) in the N-terminal domain is essential for the subunit interaction.Keywords: carbonic anhydrase 11; extracellular superoxide dismutase; heterologous expression; site-directed mutagenesis; subunit interaction; tetramer contact area Mammalian cells produce two different types of CuZnSODs, an intracellular and an extracellular form, which are almost equally efficient as catalysts and have nearly identical kinetic parameters (Tibell et al., 1987). The two enzyme forms exhibit significant sequence similarity, but they differ in several respects. The intracellular human CuZnSOD contains 153 amino acid residues per subunit, has a subunit molecular mass of 16 kDa, and is a dimer. The mature human EC-SOD subunit, on the other hand, contains 222 amino acid residues (plus an 18-residue-long signal peptide) and a N-linked oligosaccharide, resulting in a calculated molecular mass of 26.7 kDa (24.2 kDa from the polypeptide and 2.5 kDa from the oligosaccharide) (Edlund et al., 1992). The hEC-SOD protein forms a tetramer, is secreted, and binds to proteoglycans, which are found on the surface of almost every cell in the human body. The central part of the hEC-SOD sequence is homologous with the sequence of the intracellular CuZnSODs, including those amino acid residues that are metal ligands. The 48 N-terminal Reprint requests to: LenaA.E. Tibell, Department of Biochemistry, Umei University, S-901 87 Umei, Sweden; e-mail: lenat@chem.umu.se.Abbreviarions: CuZnSOD, Cu-and Zn-containing intracellular superoxide dismutase; EC-SOD, extracellular superoxide dismutase; GuHC1, guanidine hydrochloride; HCAII, human carbonic anhydrase 11: MRW, mean residue weight: SOD, superoxide dismutase. amino acid residues of hEC-SOD, and 30 of the C-terminal, have no counterparts in intracellular CuZnSODs, and no significant homology with any other sequenced protein.The three-dimensional structure of the intrace...

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“…The first moiety is a common oligomerization motif found in many proteins, the ␣-helical coiled-coil, 15 which serves to substitute for the fiber knob trimerization function. The second moiety is the target ligand that is coupled to the trimerization domain via a flexible linker peptide, such as to allow the two moieties to adopt their functional conformations.…”

Section: Introductionmentioning

confidence: 99%