A peptide corresponding to the N‐terminal 13 residues of T4 lysozyme forms an α‐helix

McLeish, Michael J.; Nielsen, Katherine J.; Wade, John D.; Craik, David J.

doi:10.1016/0014-5793(93)81187-5

Cited by 17 publications

(19 citation statements)

References 49 publications

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“…It is interesting to note that the TOCSY spectra for eledoisin in SDS showed complete NH to side-chain correlations for all residues. It is not unusual for peptide/micelle systems to give poor TOCSY spectra (McLeish et al, 1993;Rizo et al, 1993) due to the high molecular weight of the micellar aggregate, so the highquality spectra obtained for eledoisin/SDS may reflect a more facile averaging between free and micelle-bound states for eledoisin compared with other peptides.…”

Section: Resultsmentioning

confidence: 99%

A Determination of the Solution Conformation of the Nonmammalian Tachykinin Eledoisin by NMR and CD Spectroscopy

Wilson¹,

Nielsen²,

McLeish³

et al. 1994

Biochemistry

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The nonmammalian tachykinin eledoisin was investigated by use of CD and two-dimensional NMR techniques. In aqueous solution the peptide is conformationally averaged, but on addition of 50% trifluoroethanol (TFE) or sodium dodecyl sulfate (SDS) it adopts an alpha-helical structure. In TFE/H2O and SDS, residues 6-10 of eledoisin show more conformational order than the terminal regions, which undergo dynamic fraying. A possible turn in the N-terminal "address" region, the putative receptor recognition site of the peptide, is detected by NMR spectroscopy but appears to undergo substantial conformational averaging. The NMR data indicate that the helical central core of eledoisin is better defined in the micellar environment than in TFE; however, partial unfolding via 3(10) intermediates occurs in both cases. The conformational preference for SDS-bound eledoisin was examined by three-dimensional structure calculations using NMR-derived distance information in simulated annealing calculations.

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Section: Resultsmentioning

confidence: 99%

A Determination of the Solution Conformation of the Nonmammalian Tachykinin Eledoisin by NMR and CD Spectroscopy

Wilson¹,

Nielsen²,

McLeish³

et al. 1994

Biochemistry

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“…The mechanism by which micellar and/or nonmicellar SDS acts to induce/stabilize secondary structure in peptides remains poorly understood (25,30,32). However, micellar SDS is well-known to stabilize an R-helical conformation in peptides derived from helical regions of the original protein (65,69,71, and references therein). In the case of NC1(83-116), the CD spectra recorded in micellar SDS and at an SDS concentration around the critical micellar concentration (Figure 2B) revealed a helical folding.…”

Section: Discussionmentioning

confidence: 99%

Structural Analysis of the Heparin-Binding Site of the NC1 Domain of Collagen XIV by CD and NMR^,

et al. 1999

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Type XIV collagen, a fibril-associated collagen with interrupted triple helices (FACIT), interacts with the surrounding extracellular matrix and/or with cells via its binding to glycosaminoglycans (GAGs). To further characterize such interactions in the NC1 domain of chicken collagen XIV, we identified amino acids essential for heparin binding by affinity chromatography analysis after proteolytic digestion of the synthetic peptide NC1(84-116). The 3D structure of this peptide was then obtained using circular dichroism and NMR. The NC1(84-116) peptide appeared poorly structured in water, but the stabilization of its conformation by the interaction with hydrophobic surfaces or by using cosolvents (TFE, SDS) revealed a high propensity to adopt an alpha-helical folding. A 3D structure model of NC1(84-116), calculated from NMR data recorded in a TFE/water mixture, showed that the NC1-heparin binding site forms a amphipathic alpha-helix exhibiting a twisted basic groove. It is structurally similar to the consensus spatial alpha-helix model of heparin-binding [Margalit et al. (1993) J. Biol. Chem. 268, 19228-19231], except that the GAG binding domain of NC1 may be extended over 18 residues, that is, the NC1(94-111) segment. In addition, the formation of a hydrophobic groove upon helix formation suggests the contribution of additional sequences to ensure the stability of the GAG-binding domain. Overall the NC1(84-116) model exhibits a nativelike conformation which presents suitably oriented residues for the interaction with a specific GAG.

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“…Previous studies on fragments of T4 lysozyme have focused on small isolated units of secondary structure that are only structured in the presence of extremely stabilizing solvents such as trifluoroethanol (McLeish et al, 1993(McLeish et al, , 1994Najbar et al, 1995). We have taken advantage of the bipartite structure of T4 lysozyme to examine larger subdomains and the coupling between them.…”

Section: Subdomain Assembly As a Model For Foldingmentioning

confidence: 99%

Subdomain interactions as a determinant in the folding and stability of T4 lysozyme

Llinás

Marqusee

1998

Protein Science

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The folding of large, multidomain proteins involves the hierarchical assembly of individual domains. It remains unclear whether the stability and folding of small, single-domain proteins occurs through a comparable assembly of small, autonomous folding units. We have investigated the relationship between two subdomains of the protein T4 lysozyme. Thermodynamically, T4 lysozyme behaves as a cooperative unit and the unfolding transition fits a two-state model. The structure of the protein, however, resembles a dumbbell with two potential subdomains: an N-terminal subdomain (residues 13-75), and a C-terminal subdomain (residues 76-164 and 1-12). To investigate the effect of uncoupling these two subdomains within the context of the native protein, we created two circular permutations, both at the subdomain interface (residues 13 and 75). Both variants adopt an active wild-type T4 lysozyme fold. The protein starting with residue 13 is 3 kcal/mol less stable than wild type, whereas the protein beginning at residue 75 is 9 kcal/mol less stable, suggesting that the placement of the termini has a major effect on protein stability while minimally affecting the fold. When isolated as protein fragments, the C-terminal subdomain folds into a marginally stable helical structure, whereas the N-terminal subdomain is predominantly unfolded. ANS fluorescence studies indicate that, at low pH, the C-terminal subdomain adopts a loosely packed acid state. An acid state intermediate is also seen for all of the full-length variants. We propose that this acid state is comprised of an unfolded N-terminal subdomain and a loosely folded C-terminal subdomain.Keywords: circular permutation; protein folding; subdomain; T4 lysozymeThe structural elements that dictate the stability and folding of small proteins are not well understood. For large, multidomain proteins, folding appears hierarchical; individual domains are relatively autonomous with differing stabilities and independent folding (Wetlaufer, 1973;Rose, 1979;Jaenicke, 1991). Thus, the folding of these proteins involves modular assembly coupled with cooperative association. It is unclear, however, if such a modular assembly process describes the folding and stability of small, single-domain proteins.Evidence supporting the existence of modules within small, singledomain proteins has been difficult to evaluate due to the cooperative nature of proteins. The unfolding transitions of small proteins are usually well-described by the two-state (native t) unfolded; N e U) assumption, suggesting that small proteins fold as a Reprint requests to: Susan

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A peptide corresponding to the N‐terminal 13 residues of T4 lysozyme forms an α‐helix

Cited by 17 publications

References 49 publications

A Determination of the Solution Conformation of the Nonmammalian Tachykinin Eledoisin by NMR and CD Spectroscopy

A Determination of the Solution Conformation of the Nonmammalian Tachykinin Eledoisin by NMR and CD Spectroscopy

Structural Analysis of the Heparin-Binding Site of the NC1 Domain of Collagen XIV by CD and NMR^,

Subdomain interactions as a determinant in the folding and stability of T4 lysozyme

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A peptide corresponding to the N‐terminal 13 residues of T4 lysozyme forms an α‐helix

Cited by 17 publications

References 49 publications

A Determination of the Solution Conformation of the Nonmammalian Tachykinin Eledoisin by NMR and CD Spectroscopy

A Determination of the Solution Conformation of the Nonmammalian Tachykinin Eledoisin by NMR and CD Spectroscopy

Structural Analysis of the Heparin-Binding Site of the NC1 Domain of Collagen XIV by CD and NMR,

Subdomain interactions as a determinant in the folding and stability of T4 lysozyme

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Structural Analysis of the Heparin-Binding Site of the NC1 Domain of Collagen XIV by CD and NMR^,