Peter Damberg

The Alzheimer β-peptide shows temperature-dependent transitions between left-handed 31-helix, β-strand and random coil secondary structures

The temperature‐induced structural transitions of the full length Alzheimer amyloid β‐peptide [Aβ(1–40) peptide] and fragments of it were studied using CD and 1H NMR spectroscopy. The full length peptide undergoes an overall transition from a state with a prominent population of left‐handed 31 (polyproline II; PII)‐helix at 0 °C to a random coil state at 60 °C, with an average ΔH of 6.8 ± 1.4 kJ·mol−1 per residue, obtained by fitting a Zimm–Bragg model to the CD data. The transition is noncooperative for the shortest N‐terminal fragment Aβ(1–9) and weakly cooperative for Aβ(1–40) and the longer fragments. By analysing the temperature‐dependent 3JHNHα couplings and hydrodynamic radii obtained by NMR for Aβ(1–9) and Aβ(12–28), we found that the structure transition includes more than two states. The N‐terminal hydrophilic Aβ(1–9) populates PII‐like conformations at 0 °C, then when the temperature increases, conformations with dihedral angles moving towards β‐strand at 20 °C, and approaches random coil at 60 °C. The residues in the central hydrophobic (18–28) segment show varying behaviour, but there is a significant contribution of β‐strand‐like conformations at all temperatures below 20 °C. The C‐terminal (29–40) segment was not studied by NMR, but from CD difference spectra we concluded that it is mainly in a random coil conformation at all studied temperatures. These results on structural preferences and transitions of the segments in the monomeric form of Aβ may be related to the processes leading to the aggregation and formation of fibrils in the Alzheimer plaques.

Hairpin structure of a biarsenical–tetracysteine motif determined by NMR spectroscopy

The biarsenical-tetracysteine motif is a useful tag for genetic labeling of proteins with small molecules in living cells. The present study concerns the structure of a 12 amino acid peptide FLNCCPGCCMEP bound to the fluorophore ReAsH based on resorufin. (1)H NMR spectroscopy was used to determine the solution structure of the complex formed between the peptide and the ReAsH moiety. Structure calculations based on the NMR results showed that the backbone structure of the peptide is fairly well defined, with a hairpinlike turn, similar to a type-II beta-turn, formed by the central CPGC segment. The most stable complex was formed when As2 was bonded to C4 and C5 and As1 to C8 and C9. Two clear NOESY cross-peaks between the Phe1 side chain and ReAsH confirmed the close positioning of the phenyl ring of Phe1 and ReAsH. Phe1 was found to have an edge-face geometry relative to ReAsH. The close interaction between Phe1 and ReAsH may be highly significant for the fluorescence properties of the ReAsH complex.

A left-handed 31 helical conformation in the Alzheimer Aβ(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer β‐peptide is in a temperature‐dependent equilibrium between an extended left‐handed 31 helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left‐handed 31 helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Aβ(12–28) in aqueous solution at 0°C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well‐ordered secondary structure. The solvent exchange rates of the amide protons at 0°C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03–0.04 min−1) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.

Crystal structure of the P2 C-repressor: a binder of non-palindromic direct DNA repeats

As opposed to the vast majority of prokaryotic repressors, the immunity repressor of temperate Escherichia coli phage P2 (C) recognizes non-palindromic direct repeats of DNA rather than inverted repeats. We have determined the crystal structure of P2 C at 1.8 Å. This constitutes the first structure solved from the family of C proteins from P2-like bacteriophages. The structure reveals that the P2 C protein forms a symmetric dimer oriented to bind the major groove of two consecutive turns of the DNA. Surprisingly, P2 C has great similarities to binders of palindromic sequences. Nevertheless, the two identical DNA-binding helixes of the symmetric P2 C dimer have to bind different DNA sequences. Helix 3 is identified as the DNA-recognition motif in P2 C by alanine scanning and the importance for the individual residues in DNA recognition is defined. A truncation mutant shows that the disordered C-terminus is dispensable for repressor function. The short distance between the DNA-binding helices together with a possible interaction between two P2 C dimers are proposed to be responsible for extensive bending of the DNA. The structure provides insight into the mechanisms behind the mutants of P2 C causing dimer disruption, temperature sensitivity and insensitivity to the P4 antirepressor.

13C-1H NMR Relaxation and Fluorescence Anisotropy Decay Study of Tyrosine Dynamics in Motilin

Tyrosine ring dynamics of the gastrointestinal hormone motilin was studied using two independent physical methods: fluorescence polarization anisotropy decay and NMR relaxation. Motilin, a 22-residue peptide, was selectively (13)C labeled in the ring epsilon-carbons of the single tyrosine residue. To eliminate effects of differences in peptide concentration, the same motilin sample was used in both experiments. NMR relaxation rates of the tyrosine ring C(epsilon)-H(epsilon) vectors, measured at four magnetic field strengths (9.4, 11.7, 14.1, and 18.8 Tesla) were used to map the spectral density function. When the data were analyzed using dynamic models with the same number of components, the dynamic parameters from NMR and fluorescence are in excellent agreement. However, the estimated rotational correlation times depend on the choice of dynamic model. The correlation times estimated from the two-component model-free approach and the three-component models were significantly different (1.7 ns and 2.2 ns, respectively). Various earlier studies of protein dynamics by NMR and fluorescence were compared. The rotational correlation times estimated by NMR for samples with high protein concentration were on average 18% longer for folded monomeric proteins than the corresponding times estimated by fluorescence polarization anisotropy decay, after correction for differences in viscosity due to temperature and D(2)O/H(2)O ratio.