The design, synthesis, and crystallization of an alpha‐helical peptide

Eisenberg, David; Wilcox, William R.; Eshita, Steven M.; Pryciak, Peter M.; Ho, Sa V.; DeGrado, William F.

doi:10.1002/prot.340010105

Cited by 136 publications

(83 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At pH 7.5,0.15 M NaCl, 0.01 M phosphate, 23 "C, they found a AGO of -17.75 kcal/mol. The AGO of folding for melittin is also comparable to the values of -11.4 (Eisenberg et al, 1986) and -18.6 kcal/mol (Ho & DeGrado, 1987) found for designed peptides that form a-helical tetramers. The melittin tetramer is destabilized by elevating or lowering the temperature (see Fig.…”

Section: Calculation O F~g 'supporting

confidence: 76%

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Wilcox

Eisenberg

1992

Protein Science

Self Cite

View full text Add to dashboard Cite

The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an a-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 "C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pK, values of 7.5 and 8. [7][8][9][10][11][12][13][14][15][16][17][18] agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 "C depending on the pH, unfolding above and below that temperature. AC; for folding ranges from -0.085 to -0.102 cal g" K-', comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). A H o and ASo are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, w., 1959, Adv. Protein Chem.14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 "C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 "C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system. Abbreviations: CD, circular dichroism; far UV, 260-185 nm; near UV, 330-245 nm; AzgO, light absorption at 280 nm; AC:, the standard change in heat capacity from the unfolded to the folded form; At = AL -AR, where AL and AR are the absorbance of left and right circularly polarized light, respectively; AcZU, Ac at 222 nm; AtZB2, At at 282 nm; Th, a temperature at which AHo(T) = 0; T,, a temperature at gates to form an a-helical tetramer. The amount of tetwhich ASo( T ) = 0; TES, N-tris(hydroxyrnethyl)methyl-2-aminoethane sulfonic acid. Keywords

show abstract

Section: Calculation O F~g 'supporting

confidence: 76%

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Wilcox

Eisenberg

1992

Protein Science

Self Cite

View full text Add to dashboard Cite

show abstract

“…The early molten globule-like intermediates of folding proteins have a hydrophobic component to their structure (Hughson & Baldwin, 1989; Hughson et al, 1991), and the importance of the pattern of hydrophobic amino acids can be seen in the mutagenesis studies of proteins (Lim & Sauer, 1989; Kamtekar et ai., 1993). Finally, the success of the minimalist protein designs is testimony to the power of the hydrophobic pattern (Eisenberg et al, 1986;DeGrado et al, 1987DeGrado et al, , 1989; DeGrado,…”

Section: Discussionmentioning

confidence: 99%

“…Our approach to the design of a t a was to rely upon the large body of work with peptide assemblies (Lau et al, 1984;Eisenberg et al, 1986;DeGrado et al, 1987DeGrado et al, , 1989DeGrado, 1988;Mutter & Vuilleumier, 1989;O'Shea et al, 1989;Hodges et al, 1990;Mutter & Hersperger, 1990;O'Neil & DeGrado, 1990) and monomeric helices in solution (Scholtz & Baldwin, 1992). The goal was to develop a peptide system that would meet the requirements outlined above without trying to predict the structure to atomic detail.…”

Section: Rationale Goals Definition Of Success and Approachmentioning

confidence: 99%

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

Fezoui

Weaver²,

Osterhout³

1995

Protein Science

View full text Add to dashboard Cite

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...

show abstract

“…An encouraging start has been made with the preparation and structure determination of 12-and 16-residue amphipathic helices which pack to form tetrameric units (Eisenberg et al, 1986). A more ambitious undertaking, the design and synthesis of an anti-parallel 'sandwich' of , strands, has been discussed by Richardson & Richardson (1987).…”

Section: Structures Of Factitious Proteins: Inferences From Crystallomentioning

confidence: 99%

Protein engineering. The design, synthesis and characterization of factitious proteins

Shaw

1987

Biochemical Journal

View full text Add to dashboard Cite

The design, synthesis, and crystallization of an alpha‐helical peptide

Cited by 136 publications

References 34 publications

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

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

Protein engineering. The design, synthesis and characterization of factitious proteins

Contact Info

Product

Resources

About