Cyclic modular beta-sheets

Abstract

The development of peptide beta-hairpins is problematic, because folding depends on the amino acid sequence and changes to the sequence can significantly decrease folding. Robust beta-hairpins that can tolerate such changes are attractive tools for studying interactions involving protein beta-sheets and developing inhibitors of these interactions. This paper introduces a new class of peptide models of protein beta-sheets that addresses the problem of separating folding from the sequence. These model beta-sheets are macrocyclic peptides that fold in water to present a pentapeptide beta-strand along one edge; the other edge contains the tripeptide beta-strand mimic Hao [JACS 2000, 122, 7654] and two additional amino acids. The pentapeptide and Hao-containing peptide strands are connected by two delta-linked ornithine (deltaOrn) turns [JACS 2003, 125, 876]. Each deltaOrn turn contains a free alpha-amino group that permits the linking of individual modules to form divalent beta-sheets. These "cyclic modular beta-sheets" are synthesized by standard solid-phase peptide synthesis of a linear precursor followed by solution-phase cyclization. Eight cyclic modular beta-sheets 1a-1h containing sequences based on beta-amyloid and macrophage inflammatory protein 2 were synthesized and characterized by 1H NMR. Linked cyclic modular beta-sheet 2, which contains two modules of 1b, was also synthesized and characterized. 1H NMR studies show downfield alpha-proton chemical shifts, deltaOrn delta-proton magnetic anisotropy, and NOE cross-peaks that establish all compounds but 1c and 1g to be moderately or well folded into a conformation that resembles a beta-sheet. Pulsed-field gradient NMR diffusion experiments show little or no self-association at low (</=2 mM) concentrations. Changes to the residues in the Hao-containing strands of 1c and 1g improve folding and show that folding of the structures can be enhanced without altering the sequence of the pentapeptide strand. Well-folded cyclic modular beta-sheets 1a, 1b, and 1f each have a phenylalanine directly across from Hao, suggesting that cyclic modular beta-sheets containing aromatic residues across from Hao are better folded.

Figure 1

Analytical HPLC traces of (a) linear protected peptide intermediate in the synthesis of 1g, (b) unpurified cyclic modular β-sheet 1g, and (c) purified cyclic modular β-sheet 1g. Conditions: Alltech Alltima Rocket C18 column (53 × 4.5 mm), gradient of 10–90% CH₃CN in H₂O with 0.1% TFA.

Figure 2

Analytical HPLC traces of (a) purified intermediate 4, (b) unpurified linked cyclic modular β-sheet 2, and (c) purified linked cyclic modular β-sheet 2. Conditions: Alltech Alltima Rocket C18 column (53 × 4.5 mm), gradient of 10–90% CH₃CN in H₂O with 0.1% TFA.

Figure 3

600 MHz ¹H NMR spectrum of a 2 mM solution of 1a in D₂O with 50 mM CD₃CO₂D and 50 mM CD₃CO₂Na at 6 °C. Peaks marked with an asterisk (*) likely correspond to an alternate conformation.

Figure 4

ΔδH_α [δH_α(cyclic modular β-sheet)-δH_α(linear control)] values for the upper strands of the cyclic modular β-sheets.

Figure 5

Magnetic anisotropy between the diastereotopic δ-proton resonances of each ^δOrn residue in the cyclic modular β-sheets.

Figure 6

Expansions of the 600 MHz ¹H NMR spectra of 2 mM solutions of 6b, 1e, and 1f in D₂O at 6 °C showing the ^δOrn δ-proton resonances. Assignments of the diastereotopic δ-proton resonances of 1e are tentative and are based on analogy with 1f.

Figure 7

Model of a ^δOrn β-turn mimic (Ac–^δOrn–NHMe, global minimum: MacroModel v7.0; AMBER* force field; GB/SA H₂O solvent model). An arrow indicates the NOE between the H_δS and H_α.

Figure 8

Selected expansions of the ROESY spectra of 1a: (a) 2 mM in D₂O with 50 mM CD₃CO₂D and 50 mM CD₃CO₂Na at 500 MHz and 6 °C with a 200-ms spin lock time. (b) 2 mM in 9:1 H₂O/D₂O with 50 mM CD₃CO₂D and 50 mM CD₃CO₂Na at 600 MHz and 6 °C with a 200-ms spin lock time and gradient water suppression.

Figure 9

Key NOEs shown in the ROESY spectra of cyclic modular β-sheets 1a, 1b, 1d, 1e, 1f, and 1h in D₂O.

Figure 10

Interstrand NH–NH and H_α–NH NOEs in the 800 MHz NOESY spectra of 1a and/or 1b in 9:1 H₂O/D₂O. The hydrazide NH groups do not show up under these conditions and do not give NOEs.

Figure 11

Diffusion coefficient of 1a as a function of concentration. The diffusion coefficient was measured by 800 MHz PFG NMR diffusion experiments at 6 °C in D₂O in a buffer of 50 mM CD₃COOD and 50 mM CD₃COONa. Each value represents an average measured for three resonances. Error bars are the standard deviations of these measurements.

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