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Packed protein bilayers in the 0.90 A resolution structure of a designed alpha helical bundle.

ABSTRACT: A 12-residue peptide designed to form an alpha-helix and self-associate into an antiparallel 4-alpha-helical bundle yields a 0.9 A crystal structure revealing unanticipated features. The structure was determined by direct phasing with the "Shake-and-Bake" program, and contains four crystallographically distinct 12-mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal alpha-helices hydrogen bonded head-to-tail into columns, which in turn pack side-by-side into sheets spanning the width of the crystal. Within each sheet, the alpha-helices run antiparallel and are closely spaced (9-10 A center-to-center). The sheets are more loosely packed against each other (13-14 A between helix centers). Each sheet is amphiphilic: apolar leucine side chains project from one face, charged lysine and glutamate side chains from the other face. The sheets are stacked with two polar faces opposing and two apolar faces opposing. The result is a periodic biomaterial composed of packed protein bilayers, with alternating polar and apolar interfaces. All of the 30 water molecules in the unit cell lie in the polar interface or between the stacked termini of helices. A section through the sheet reveals that the helices packed at the apolar interface resemble the four-alpha-helical bundle of the design, but the helices overhang parts of the adjacent bundles, and the helix crossing angles are less steep than intended (7-11 degrees rather than 18 degrees).

SUBMITTER: Prive GG

PROVIDER: S-EPMC2144382 | biostudies-other | 1999 Jul

REPOSITORIES: biostudies-other

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Packed protein bilayers in the 0.90 A resolution structure of a designed alpha helical bundle.

Privé G G GG Anderson D H DH Wesson L L Cascio D D Eisenberg D D

Protein science : a publication of the Protein Society 19990701 7

A 12-residue peptide designed to form an alpha-helix and self-associate into an antiparallel 4-alpha-helical bundle yields a 0.9 A crystal structure revealing unanticipated features. The structure was determined by direct phasing with the "Shake-and-Bake" program, and contains four crystallographically distinct 12-mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal alpha-helices hydrogen bonded head-to-tail into columns, which in turn pack side-by ...[more]

PMID: 10422828

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Project description:The primary and secondary coordination spheres of metal binding sites in metalloproteins have been investigated extensively, leading to the creation of high-performing functional metalloproteins; however, the impact of the overall structure of the protein scaffold on the unique properties of metalloproteins has rarely been studied. A primary example is the binuclear CuA center, an electron transfer cupredoxin domain of photosynthetic and respiratory complexes and, recently, a protein coregulated with particulate methane and ammonia monooxygenases. The redox potential, Cu-Cu spectroscopic features, and a valence delocalized state of CuA are difficult to reproduce in synthetic models, and every artificial protein CuA center to-date has used a modified cupredoxin. Here, we present a fully functional CuA center designed in a structurally nonhomologous protein, cytochrome c peroxidase (CcP), by only two mutations (CuACcP). We demonstrate with UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopy that CuACcP is valence delocalized. Continuous wave and pulsed (HYSCORE) X-band EPR show it has a highly compact gz area and small Az hyperfine principal value with g and A tensors that resemble axially perturbed CuA. Stopped-flow kinetics found that CuA formation proceeds through a single T2Cu intermediate. The reduction potential of CuACcP is comparable to native CuA and can transfer electrons to a physiological redox partner. We built a structural model of the designed Cu binding site from extended X-ray absorption fine structure spectroscopy and validated it by mutation of coordinating Cys and His residues, revealing that a triad of residues (R48C, W51C, and His52) rigidly arranged on one α-helix is responsible for chelating the first Cu(II) and that His175 stabilizes the binuclear complex by rearrangement of the CcP heme-coordinating helix. This design is a demonstration that a highly conserved protein fold is not uniquely necessary to induce certain characteristic physical and chemical properties in a metal redox center.

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Packed protein bilayers in the 0.90 A resolution structure of a designed alpha helical bundle.

Publications

Packed protein bilayers in the 0.90 A resolution structure of a designed alpha helical bundle.

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