Unfolding Simulations of Holomyoglobin from Four Mammals: Identification of Intermediates and β-Sheet Formation from Partially Unfolded States

Myoglobin (Mb) is a centrally important, widely studied mammalian protein. While much work has investigated multi-step unfolding of apoMb using acid or denaturant, holomyoglobin unfolding is poorly understood despite its biological relevance. We present here the first systematic unfolding simulations of holoMb and the first comparative study of unfolding of protein orthologs from different species (sperm whale, pig, horse, and harbor seal). We also provide new interpretations of experimental mean molecular ellipticities of myoglobin intermediates, notably correcting for random coil and number of helices in intermediates. The simulated holoproteins at 310 K displayed structures and dynamics in agreement with crystal structures (R g ~1.48-1.51 nm, helicity ~75%). At 400 K, heme was not lost, but some helix loss was observed in pig and horse, suggesting that these helices are less stable in terrestrial species. At 500 K, heme was lost within 1.0-3.7 ns. All four proteins displayed exponentially decaying helix structure within 20 ns. The C- and F-helices were lost quickly in all cases. Heme delayed helix loss, and sperm whale myoglobin exhibited highest retention of heme and D/E helices. Persistence of conformation (RMSD), secondary structure, and ellipticity between 2-11 ns was interpreted as intermediates of holoMb unfolding in all four species. The intermediates resemble those of apoMb notably in A and H helices, but differ substantially in the D-, E- and F-helices, which interact with heme. The identified mechanisms cast light on the role of metal/cofactor in poorly understood holoMb unfolding. We also observed β-sheet formation of several myoglobins at 500 K as seen experimentally, occurring after disruption of helices to a partially unfolded, globally disordered state; heme reduced this tendency and sperm-whale did not display any sheet propensity during the simulations

An improved approach to steady-state analysis of monoamine oxidases

The search for new monoamine oxidaseinhibitors aims to identify potential lead compounds thatare more potent and selective than current drugs for use intreating a variety of neuropsychiatric and neurodegenerativeconditions. An integral part of this process is a kineticexamination of monoamine oxidases in the presence of theinhibitor, to determine potency and selectivity and toobtain information on mechanism. To date, kinetic dataobtained with a probe substrate have been analysed byfitting to the Michaelis–Menten equation which describes aunireactant process in which velocity is related to substrateconcentration in a rectangular hyperbolic manner. In thisstudy, we present evidence that monoamine oxidaseactivity is often not adequately described by this approach.We outline a novel equation strategy that takes account ofsubstrate and inhibitor binding to oxidised and reducedenzyme forms, and quantifies differences between substratesand inhibitors in this regard. When combined withplate reader-based experimental techniques that allow largenumbers of substrate and inhibitor concentrations to beused, and the global nonlinear regression facilities ofGraphPad Prism software, this straightforward approachallows more appropriate analyses of monoamine oxidaseby non-experts than has previously been possible.Keywords Monoamine oxidase Steady-state kinetics Equations Inhibition GraphPad PrismIntroductionMonoamine oxidases (MAOs) are targets for therapeuticsdesigned to treat numerous neuropsychiatric and neurodegenerativeconditions (Youdim et al. 2006). To date,drugs targeting MAOs have been developed as inhibitorsof enzymatic activity, designed to increase the availabilityof biogenic amine transmitters and neuromodulators,to decrease formation of reactive oxygen speciesand also for in vitro use to probe mechanisms of MAOcatalysis. Preclinical development and experimental useof MAO inhibitors require determination of inhibitorpotency and mechanism in steady-state assays of enzymeactivity. Such assays typically involve inclusion of theinhibitor, at several concentrations, in assays of MAOactivity versus a range of substrate concentrationsextending either side of the KM value. Historically, initialrate data have then been plotted and analysed by fittingto a hyperbolic or linearised version of the Michaelis–Menten equation. Analyses are rapid and easy to do withregression software, and interpretation of results appearsquit

Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering

We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and quaternary conformational changes of human hemoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. We also report data on optically induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide applicability of the technique. By providing insights into the structural dynamics of proteins functioning in their natural environment, TR-WAXS complements and extends results obtained with time-resolved optical spectroscopy and X-ray crystallography