8 research outputs found
Mapping local structural perturbations in the native state of stefin B (cystatin B) under amyloid forming conditions
Unlike a number of amyloid-forming proteins, stefins, and in particular stefin B (cystatin B) form amyloids under conditions where the native state predominates. In order to trigger oligomerization processes, the stability of the protein needs to be compromised, favoring structural re-arrangement however, accelerating fibril formation is not a simple function of protein stability. We report here on how optimal conditions for amyloid formation lead to the destabilization of dimeric and tetrameric states of the protein in favor of the monomer. Small, highly localized structural changes can be mapped out that allow us to visualize directly areas of the protein which eventually become responsible for triggering amyloid formation. These regions of the protein overlap with the Cu (II)-binding sites which we identify here for the first time. We hypothesize that in vivo modulators of amyloid formation may act similarly to painstakingly optimized solvent conditions developed in vitro. We discuss these data in the light of current structural models of stefin B amyloid fibrils based on H-exchange data, where the detachment of the helical part and the extension of loops were observed
Equatorial active site compaction and electrostatic reorganization in catechol-O-methyltransferase
Catechol-O-methyltransferase (COMT) is a model
S-adenosyl-l-methionine (SAM) dependent methyl transferase,
which catalyzes the methylation of catecholamine neurotransmitters
such as dopamine in the primary pathway of neurotransmitter deactivation
in animals. Despite extensive study, there is no consensus view of
the physical basis of catalysis in COMT. Further progress requires
experimental data that directly probes active site geometry, protein
dynamics and electrostatics, ideally in a range of positions along
the reaction coordinate. Here we establish that sinefungin, a fungal-derived
inhibitor of SAM-dependent enzymes that possess transition state-like
charge on the transferring group, can be used as a transition state
analog of COMT when combined with a catechol. X-ray crystal structures
and NMR backbone assignments of the ternary complexes of the soluble
form of human COMT containing dinitrocatechol, Mg2+ and
SAM or sinefungin were determined. Comparison and further analysis
with the aid of density functional theory calculations and molecular
dynamics simulations provides evidence for active site ācompactionā,
which is driven by electrostatic stabilization between the transferring
methyl group and āequatorialā active site residues that
are orthogonal to the donorāacceptor (pseudo reaction) coordinate.
We propose that upon catecholamine binding and subsequent proton transfer
to Lys 144, the enzyme becomes geometrically preorganized, with little
further movement along the donorāacceptor coordinate required
for methyl transfer. Catalysis is then largely facilitated through
stabilization of the developing charge on the transferring methyl
group via āequatorialā H-bonding and electrostatic interactions
orthogonal to the donorāacceptor coordinate
Real-time pure shift N-15 HSQC of proteins: a real improvement in resolution and sensitivity
Spectral resolution in proton NMR spectroscopy is reduced by the splitting of resonances into multiplets due to the effect of homonuclear scalar couplings. Although these effects are often hidden in protein NMR spectroscopy by low digital resolution and routine apodization, behind the scenes homonuclear scalar couplings increase spectral overcrowding. The possibilities for biomolecular NMR offered by new pure shift NMR methods are illustrated here. Both resolution and sensitivity are improved, without any increase in experiment time. In these experiments, free induction decays are collected in short bursts of data acquisition, with durations short on the timescale of J-evolution, interspersed with suitable refocusing elements. The net effect is real-time (t 2) broadband homodecoupling, suppressing the multiplet structure caused by protonāproton interactions. The key feature of the refocusing elements is that they discriminate between the resonances of active (observed) and passive (coupling partner) spins. This can be achieved either by using band-selective refocusing or by the BIRD element, in both cases accompanied by a nonselective 180Ā° proton pulse. The latter method selects the active spins based on their one-bond heteronuclear J-coupling to 15N, while the former selects a region of the 1H spectrum. Several novel pure shift experiments are presented, and the improvements in resolution and sensitivity they provide are evaluated for representative samples: the N-terminal domain of PGK; ubiquitin; and two mutants of the small antifungal protein PAF. These new experiments, delivering improved sensitivity and resolution, have the potential to replace the current standard HSQC experiments
Modulation of contact order effects in the two-state folding of stefins A and B.
It is well established that contact order and folding rates are correlated for small proteins. The folding rates of stefins A and B differ by nearly two orders of magnitude despite sharing an identical native fold and hence contact order. We break down the determinants of this behavior and demonstrate that the modulation of contact order effects can be accounted for by the combined contributions of a framework-like mechanism, characterized by intrinsic helix stabilities, together with nonnative helical backbone conformation and nonnative hydrophobic interactions within the folding transition state. These contributions result in the formation of nonnative interactions in the transition state as evidenced by the opposing effects on folding rate and stability of these proteins