The origin of the stability of isolated β-hairpins in aqueous
solution is unclear with contrasting
opinions as to the relative importance of interstrand hydrogen bonding,
hydrophobic interactions, and
conformational preferences, the latter being associated largely with
the turn sequence. We have designed an
unconstrained 16-residue peptide that we show folds autonomously in
water to form a β-hairpin that mimics
the two-stranded anti-parallel β-sheet DNA binding motif of the
met repressor dimer. The designed peptide,
with a type I‘ turn (INGK), is shown by CD and a range of NMR
parameters to be appreciably folded (≈50%
at 303 K) in aqueous solution with the predicted alignment of the
peptide backbone. We show that the folding
transition approximates to a two-state model. The hairpin has a
marked temperature-dependent stability, reaching
a maximum value at 303 K in water with both lower and higher
temperatures destabilizing the folded structure.
Van't Hoff analysis of Hα chemical shifts, reveals that folding
is endothermic and entropy-driven in aqueous
solution with a large negative ΔC
p, all of
which are reminiscent of proteins with hydrophobic cores,
pointing
to the hydrophobic effect as the dominant stabilizing interaction in
water. We have examined the conformational
properties of the C-terminal β-strand (residues 9−16) in isolation
and have shown that
3
J
α
N values and
backbone
intra- and inter-residue Hα-NH NOE intensities deviate from those
predicted for a random coil, indicating
that the β-strand has a natural predisposition to adopt an extended
conformation in the absence of secondary
structure interactions. A family of β-hairpin structures
calculated from 200 (distance and torsion angle)
restraints
using molecular dynamics shows that the conformation of the hairpin
mimics closely the DNA binding face
of the met repressor dimer (backbone RMSD between
corresponding β-strands of 1.0 ± 0.2 Å).
The extracellular siderophore from Mycobacterium smegmatis, exochelin MS, was isolated from iron-deficiently grown cultures and purified to > 98% by a combination of ion-exchange chromatography and h.p.l.c. The material is unextractable into organic solvents, is basic (pI = 9.3-9.5), has a lambda max at 420 nm and a probable Ks for Fe3+ of between 10(25) and 10(30). Its structure has been determined by examination of desferri- and ferri-exochelin and its gallium complex. The methods used were electrospray-m.s. and one- and two-dimensional (NOESY, DQF-COSY and TOCSY) 1H n.m.r. The constituent amino acids were examined by chiral g.l.c analysis of N-trifluoroacetyl isopropyl and N-pentafluoropropionyl methyl esters after hydrolysis, and reductive HI hydrolysis, of the siderophore. The exochelin is a formylated pentapeptide: N-(delta-N-formyl,delta N-hydroxy-R-ornithyl) -beta-alaninyl-delta N-hydroxy-R-ornithinyl-R-allo-threoninyl-delta N-hydroxy-S-ornithine. The linkages involving the three ornithine residues are via their delta N(OH) and alpha-CO groups leaving three free alpha-NH2 groups. Although there are two peptide bonds, these involve the three R (D)-amino acids. Thus the molecule has no conventional peptide bond, and this suggests that it will be resistant to peptidase hydrolysis. The co-ordination centre with Fe3+ is hexadenate in an octahedral structure involving the three hydroxamic acid groups. Molecular modelling shows it to have similar features to other ferric trihydroxamate siderophores whose three-dimensional structures have been established. The molecule is shown to have little flexibility around the iron chelation centre, although the terminal (Orn-3) residue, which is not involved in iron binding except at its delta N atom, has more motional freedom.
Exochelin MN has some structural features in common with other siderophores, but has a unique three-dimensional structure, which is presumably important for its specific activity in M. leprae. Exochelin MN may be a target for drug design in the fight against infection with this pathogen.
The solution structure of the dodecamer duplex d(CTTTTGCAAAAG)(2)and its 2:1 complex with the bis -benzimidazole Hoechst 33258 has been investigated by NMR and NOE-restrained molecular dynamics (rMD) simulations. Drug molecules are bound in each of the two A-tracts with the bulky N-methylpiperazine ring of each drug located close to the central TG (CA) step, binding essentially to the narrow minor groove of each A-tract. MD simulations over 1 ns, using an explicit solvation model, reveal time-averaged sequence-dependent narrowing of the minor groove from the 3'-end towards the 5'-end of each TTTT sequence. Distinct junctions at the TpG (CpA) steps, characterised by large positive roll, low helical and propeller twists and rapid AT base pair opening rates, add to the widening of the groove at these sites and appear to account for the bound orientation of the two drug molecules with the N-methylpiperazine ring binding in the wider part of the groove close to the junctions. Comparisons between the free DNA structure and the 2:1 complex (heavy atom RMSD 1.55 A) reveal that these sequence-dependent features persist in both structures. NMR studies of the sequence d(GAAAAGCTTTTC)(2), in which the A-tracts have been inverted with the elimination of the TpG junctions, results in loss of orientational specificity of Hoechst 33258 and formation of multiple bound species in solution, consistent with the drug binding in a number of different orientations.
We present reference data and a javascript web page which allow the rapid identification and quantification of residual solvents by NMR. The data encompass all of the ICH-prescribed solvents and were obtained for a number of NMR solvents. We also present an example of its application.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.