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

Abstract

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.

Elucidation of the structural stability and dynamics of heterogeneous intermediate ensembles in unfolding pathway of the N-terminal domain of TDP-43

“…30,34 Therefore, high-temperature MD simulations have been employed to investigate the unfolding pathways or stabilities of several proteins. [35][36][37][38][39] To increase the rate of the unfolding process, both experimental and computational methods utilize high temperature, high pressure, low pH, or chemical denaturants (guanidinium chloride or urea). In the present work, we have employed allatom MD simulations to obtain detailed insights into the unfolding and thermostability of the NTD in 8 M urea at 300 K, 350 K, 400 K, 450 K and 500 K. The results of the cumulative microsecond simulations indicate (i) the presence of stable intermediates (I N and I U ) and the transition state (TS) along the unfolding pathway; (ii) the formation of a molten globule (MG) during an early unfolding intermediate, I N ; (iii) conformationally heterogeneous I U stabilized by non-native contacts and hydrogen bonds; (iv) initiation of unfolding through disruption of b3; and (v) the presence of residual structures in the unfolded state.…”

New supercharging reagents produce highly charged protein ions in native mass spectrometry

“…55, 56 The percentage of apo-myoglobin observed in all spectra in Figure 3 as a function of the average charge of all myoglobin species in a given mass spectrum is shown in Figure 4. These data show a trend of increasing fraction of apo-myoglobin with increasing charging obtained with the supercharging reagents.…”

Force-Field Benchmarking by Alternatives: A Systematic Study of Ten Small α- and β-Proteins

“…The sequence-structure relationship is evidently context dependent, and changes in the environment, e.g. heating or membrane interaction, commonly induce secondary structure changes [27][28][29] ; it is also well-known that salt and pH can affect the structure of a sequence 30,31 , and even very similar sequences can in some cases feature quite distinct structures 32 . Adding to this complexity is the structure biases of modern protein force fields, which may predict another structure than the one seen experimentally 14,25,33,34 .…”