A pH-dependent conformational transition of Abeta peptide and physicochemical properties of the conformers in the glial cell.
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ABSTRACT:
SUBMITTER: Matsunaga Y
PROVIDER: S-EPMC1222337 | biostudies-other | 2002 Feb
REPOSITORIES: biostudies-other
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Project description:The matrix-2 (M2) protein from influenza A virus is a tetrameric, integral transmembrane (TM) protein that plays a vital role in viral replication by proton flux into the virus. The His37 tetrad is a pH sensor in the center of the M2 TM helix that activates the channel in response to the low endosomal pH. M2 consists of different regions that are believed to be involved in membrane targeting, packaging, nucleocapsid binding, and proton transport. Although M2 has been the target of many experimental and theoretical studies that have led to significant insights into its structure and function under differing conditions, the main mechanism of proton transport, its conformational dynamics, and the role of the amphipathic helices (AHs) on proton conductance remain elusive. To this end, we have applied explicit solvent constant pH molecular dynamics using the multisite ?-dynamics approach (CpHMDMS?D) to investigate the buried ionizable residues comprehensively and to elucidate their effect on the conformational transition. Our model recapitulates the pH-dependent conformational transition of M2 from closed to open state when the AH domain is included in the M2 construct, revealing the role of the amphipathic helices on this transition and shedding light on the proton-transport mechanism. This work demonstrates the importance of including the amphipathic helices in future experimental and theoretical studies of ion channels. Finally, our work shows that explicit solvent CpHMDMS?D provides a realistic pH-dependent model for membrane proteins.
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Project description:A recently engineered mutant of cyan fluorescent protein (WasCFP) that exhibits pH-dependent absorption suggests that its tryptophan-based chromophore switches between neutral (protonated) and charged (deprotonated) states depending on external pH. At pH 8.1, the latter gives rise to green fluorescence as opposed to the cyan color of emission that is characteristic for the neutral form at low pH. Given the high energy cost of deprotonating the tryptophan at the indole nitrogen, this behavior is puzzling, even if the stabilizing effect of the V61K mutation in proximity to the protonation/deprotonation site is considered. Because of its potential to open new avenues for the development of optical sensors and photoconvertible fluorescent proteins, a mechanistic understanding of how the charged state in WasCFP can possibly be stabilized is thus important. Attributed to the dynamic nature of proteins, such understanding often requires knowledge of the various conformations adopted, including transiently populated conformational states. Transient conformational states triggered by pH are of emerging interest and have been shown to be important whenever ionizable groups interact with hydrophobic environments. Using a combination of the weighted-ensemble sampling method and explicit-solvent constant pH molecular dynamics (CPHMD(MS?D)) simulations, we have identified a solvated transient state, characterized by a partially open ?-barrel where the chromophore pKa of 6.8 is shifted by over 20 units from that of the closed form (6.8 and 31.7, respectively). This state contributes a small population at low pH (12% at pH 6.1) but becomes dominant at mildly basic conditions, contributing as much as 53% at pH 8.1. This pH-dependent population shift between neutral (at pH 6.1) and charged (at pH 8.1) forms is thus responsible for the observed absorption behavior of WasCFP. Our findings demonstrate the conditions necessary to stabilize the charged state of the WasCFP chromophore (namely, local solvation at the deprotonation site and a partial flexibility of the protein ?-barrel structure) and provide the first evidence that transient conformational states can control optical properties of fluorescent proteins.
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Project description:The transition from ?-helical to ?-hairpin conformations of ?-syn12 peptide is characterized here using long timescale, unbiased molecular dynamics (MD) simulations in explicit solvent models at physiological and acidic pH values. Four independent normal MD trajectories, each 2500 ns, are performed at 300 K using the GROMOS 43A1 force field and SPC water model. The most clustered structures at both pH values are ?-hairpin but with different turns and hydrogen bonds. Turn9-6 and four hydrogen bonds (HB9-6, HB6-9, HB11-4 and HB4-11) are formed at physiological pH; turn8-5 and five hydrogen bonds (HB8-5, HB5-8, HB10-3, HB3-10 and HB12-1) are formed at acidic pH. A common folding mechanism is observed: the formation of the turn is always before the formation of the hydrogen bonds, which means the turn is always found to be the major determinant in initiating the transition process. Furthermore, two transition paths are observed at physiological pH. One of the transition paths tends to form the most-clustered turn and improper hydrogen bonds at the beginning, and then form the most-clustered hydrogen bonds. Another transition path tends to form the most-clustered turn, and turn5-2 firstly, followed by the formation of part hydrogen bonds, then turn5-2 is extended and more hydrogen bonds are formed. The transition path at acidic pH is as the same as the first path described at physiological pH.
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Project description:The amyloid beta-peptides (Abetas), containing 39-43 residues, are the key protein components of amyloid deposits in Alzheimer's disease. To structurally characterize the dynamic behavior of Abeta(40), 12 independent long-time molecular dynamics (MD) simulations for a total of 850 ns were performed on both the wide-type peptide and its mutant in both aqueous solution and a biomembrane environment. In aqueous solution, an alpha-helix to beta-sheet conformational transition for Abeta(40) was observed, and an entire unfolding process from helix to coil was traced by MD simulation. Structures with beta-sheet components were observed as intermediates in the unfolding pathway of Abeta(40). Four glycines (G(25), G(29), G(33), and G(37)) are important for Abeta(40) to form beta-sheet in aqueous solution; mutations of these glycines to alanines almost abolished the beta-sheet formation and increased the content of the helix component. In the dipalmitoyl phosphatidylcholine (DPPC) bilayer, the major secondary structure of Abeta(40) is a helix; however, the peptide tends to exit the membrane environment and lie down on the surface of the bilayer. The dynamic feature revealed by our MD simulations rationalized several experimental observations for Abeta(40) aggregation and amyloid fibril formation. The results of MD simulations are beneficial to understanding the mechanism of amyloid formation and designing the compounds for inhibiting the aggregation of Abeta and amyloid fibril formation.
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Project description:X-ray structures of carbonmonoxymyoglobin (MbCO) are available for different pH values. We used conventional electrostatic continuum methods to calculate the titration behavior of MbCO in the pH range from 3 to 7. For our calculations, we considered five different x-ray structures determined at pH values of 4, 5, and 6. We developed a Monte Carlo method to sample protonation states and conformations at the same time so that we could calculate the population of the considered MbCO structures at different pH values and the titration behavior of MbCO for an ensemble of conformers. To increase the sampling efficiency, we introduced parallel tempering in our Monte Carlo method. The calculated population probabilities show, as expected, that the x-ray structures determined at pH 4 are most populated at low pH, whereas the x-ray structure determined at pH 6 is most populated at high pH, and the population of the x-ray structures determined at pH 5 possesses a maximum at intermediate pH. The calculated titration behavior is in better agreement with experimental results compared to calculations using only a single conformation. The most striking feature of pH-dependent conformational changes in MbCO-the rotation of His-64 out of the CO binding pocket-is reproduced by our calculations and is correlated with a protonation of His-64, as proposed earlier.
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Project description:Glycinamide ribonucleotide transformylase (GART) undergoes a pH-dependent coil-helix transition with pK(a) approximately 7. An alpha-helix is formed at high pH spanning 8 residues of a 21-residue-long loop, comprising the segment Thr120-His121-Arg122-Gln123-Ala124-Leu125-Glu126-Asn127. To understand the electrostatic nature of this loop-helix, called the activation loop-helix, which leads to the formation and stability of the alpha-helix, pK(a) values of all ionizable residues of GART have been calculated, using Poisson-Boltzmann electrostatic calculations and crystallographic data. Crystallographic structures of high and low pH E70A GART have been used in our analysis. Low pK(a) values of 5.3, 5.3, 3.9, 1.7, and 4.7 have been calculated for five functionally important histidines, His108, His119, His121, His132, and His137, respectively, using the high pH E70A GART structure. Ten theoretical single and double mutants of the high pH E70A structure have been constructed to identify pairwise interactions of ionizable residues, which have aided in elucidating the multiplicity of electrostatic interactions of the activation loop-helix, and the impact of the activation helix on the catalytic site. Based on our pK(a) calculations and structural data, we propose that: (1) His121 forms a molecular switch for the coil-helix transition of the activation helix, depending on its protonation state; (2) a strong electrostatic interaction between His132 and His121 is observed, which can be of stabilizing or destabilizing nature for the activation helix, depending on the relative orientation and protonation states of the rings of His121 and His132; (3) electrostatic interactions involving His119 and Arg122 play a role in the stability of the activation helix; and (4) the activation helix contains the helix-promoting sequence Arg122-Gln123-Ala124-Leu125-Glu126, but its alignment relative to the N and C termini of the helix is not optimal, and is possibly of a destabilizing nature. Finally, we provide electrostatic evidence that the formation and closure of the activation helix create a hydrophobic environment for catalytic-site residue His108, to facilitate catalysis.
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Project description:The bacterial K+ channel KcsA from Streptomyces lividans was analyzed by neutron and x-ray small-angle solution scattering. The C-terminally truncated version of KcsA, amenable to crystallographic studies, was compared with the full-length channel. Analyzing the scattering data in terms of radius of gyration reveals differences between both KcsA species of up to 13.2 A. Equally, the real-space distance distribution identifies a 40 to 50 A extension of full-length KcsA compared to its C-terminally truncated counterpart. We show that the x-ray and neutron scattering data are amenable for molecular shape reconstruction of full-length KcsA. The molecular envelopes calculated display an hourglass-shaped structure within the C-terminal intracellular domain. The C-terminus extends the membrane spanning region of KcsA by 54-70 A, with a central constriction 10-30 A wide. Solution scattering techniques were further employed to characterize the KcsA channel under acidic conditions favoring its open conformation. The full-length KcsA at pH 5.0 shows the characteristics of a dumbbell-shaped macromolecular structure, originating from dimerization of the tetrameric K+ channel. Since C-terminally truncated KcsA measured under the same low pH conditions remains tetrameric, oligomerization of full-length KcsA seems to proceed via structurally changed C-terminal domains. The determined maximum dimensions of the newly formed complex increase by 50-60%. Shape reconstruction of the pseudooctameric complex indicates the pH-induced conformational reorganization of the intracellular C-terminal domain.
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