Sevoflurane-induced structural changes in a four-alpha-helix bundle protein

The mechanisms whereby volatile general anesthetics reversibly alter protein function in the central nervous system remain obscure. Using three different spectroscopic approaches, evidence is presented that binding of the modern general anesthetic sevoflurane to the hydrophobic core of a model four-alpha-helix bundle protein results in structural changes. Aromatic residues in the hydrophobic core reorient into new environments upon anesthetic binding, and the protein as a whole becomes less dynamic and exhibits structural tightening. Comparable structural changes in the predicted in vivo protein targets, such as the gamma-aminobutyric acid type A receptor and the N-methyl-D-aspartate receptor, may underlie some, or all, of the behavioral effects of these widely used clinical agents.     

Expression and characterization of a four-alpha-helix bundle protein that binds the volatile general anesthetic halothane

The structural features of volatile anesthetic binding sites on proteins are being investigated with the use of a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. The current study describes the bacterial expression, purification, and initial characterization of the four-alpha-helix bundle (Aalpha(2)-L1M/L38M)(2). The alpha-helical content and stability of the expressed protein are comparable to that of the chemically synthesized four-alpha-helix bundle (Aalpha(2)-L38M)(2) reported earlier. The affinity for binding halothane is somewhat improved with a K(d) = 120 +/- 20 microM as determined by W15 fluorescence quenching, attributed to the L1M substitution. Near-UV circular dichroism spectroscopy demonstrated that halothane binding changes the orientation of the aromatic residues in the four-alpha-helix bundle. Nuclear magnetic resonance experiments reveal that halothane binding results in narrowing of the peaks in the amide region of the one-dimensional proton spectrum, indicating that bound anesthetic limits protein dynamics. This expressed protein should prove to be amenable to nuclear magnetic resonance structural studies on the anesthetic complexes, because of its relatively small size (124 residues) and the high affinities for binding volatile anesthetics. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules and will provide guidelines regarding the general architecture of binding sites on central nervous system proteins.     

Fluorous effect in proteins: de novo design and characterization of a four-alpha-helix bundle protein containing hexafluoroleucine

Several studies have demonstrated that proteins incorporating fluorinated analogues of hydrophobic amino acids such as leucine and valine into their hydrophobic cores exhibit increased stability toward thermal denaturation and unfolding by guanidinium chloride. However, estimates for the increase in the thermodynamic stability of a protein (DeltaDeltaG(unfold)) afforded by the substitution of a hydrophobic amino acid with its fluorinated analogue vary quite significantly. To address this, we have designed a peptide that adopts an antiparallel four-helix bundle structure in which the hydrophobic core is packed with leucine, and investigated the effects of substituting the central two layers of the core with L-5,5,5,5',5',5'-hexafluoroleucine (hFLeu). We find that DeltaDeltaG(unfold) is increased by 0.3 kcal/mol per hFLeu residue. This is in good agreement with the predicted increase in DeltaDeltaG(unfold) of 0.4 kcal/mol per residue arising from the increased hydrophobicity of the hFLeu side chain, which we determined experimentally from partitioning measurements on hFLeu and leucine. The increased stability of this fluorinated protein may therefore be ascribed to simple hydrophobic effects, rather than specific "fluorous" interactions between the hFLeu residues.     

General trends of dihedral conformational transitions in a globular protein

Dihedral conformational transitions are analyzed systematically in a model globular protein, cytochrome P450cam, to examine their structural and chemical dependences through combined conventional molecular dynamics (cMD), accelerated molecular dynamics (aMD) and adaptive biasing force (ABF) simulations. The aMD simulations are performed at two acceleration levels, using dihedral and dual boost, respectively. In comparison with cMD, aMD samples protein dihedral transitions approximately two times faster on average using dihedral boost, and ～3.5 times faster using dual boost. In the protein backbone, significantly higher dihedral transition rates are observed in the bend, coil, and turn flexible regions, followed by the β bridge and β sheet, and then the helices. Moreover, protein side chains of greater length exhibit higher transition rates on average in the aMD‐enhanced sampling. Side chains of the same length (particularly N_χ = 2) exhibit decreasing transition rates with residues when going from hydrophobic to polar, then charged and aromatic chemical types. The reduction of dihedral transition rates is found to be correlated with increasing energy barriers as identified through ABF free energy calculations. These general trends of dihedral conformational transitions provide important insights into the hierarchical dynamics and complex free energy landscapes of functional proteins. Proteins 2016; 84:501–514. © 2016 Wiley Periodicals, Inc.     

Heme redox potential control in de novo designed four-alpha-helix bundle proteins

The effects of various mechanisms of metalloporphyrin reduction potential modulation were investigated experimentally using a robust, well-characterized heme protein maquette, synthetic protein scaffold H10A24 [?CH(3)()CONH-CGGGELWKL.HEELLKK.FEELLKL.AEERLKK. L-CONH(2)()?(2)](2). Removal of the iron porphyrin macrocycle from the high dielectric aqueous environment and sequestration within the hydrophobic core of the H10A24 maquette raises the equilibrium reduction midpoint potential by 36-138 mV depending on the hydrophobicity of the metalloporphyrin structure. By incorporating various natural and synthetic metalloporphyrins into a single protein scaffold, we demonstrate a 300-mV range in reduction potential modulation due to the electron-donating/withdrawing character of the peripheral macrocycle substituents. Solution pH is used to modulate the metalloporphyrin reduction potential by 160 mV, regardless of the macrocycle architecture, by controlling the protonation state of the glutamate involved in partial charge compensation of the ferric heme. Attempts to control the reduction potential by inserting charged amino acids into the hydrophobic core at close proximity to the metalloporphyrin lead to varied success, with H10A24-L13E lowering the E(m8.5) by 40 mV, H10A24-E11Q raising it by 50 mV, and H10A24-L13R remaining surprisingly unaltered. Modifying the charge of the adjacent metalloporphyrin, +1 for iron(III) protoporphyrin IX or neutral for zinc(II) protoporphyrin IX resulted in a loss of 70 mV [Fe(III)PPIX](+) - [Fe(III)PPIX](+) interaction observed in maquettes. Using these factors in combination, we illustrate a 435-mV variation of the metalloporphyrin reduction midpoint potential in a simple heme maquette relative to the about 800-mV range observed for natural cytochromes. Comparison between the reduction potentials of the heme maquettes and other de novo designed heme proteins reveals global trends in the E(m) values of synthetic cytochromes.     

Role of aromatic side chains in the binding of volatile general anesthetics to a four-alpha-helix bundle

Currently, the mechanism by which anesthesia occurs is thought to involve the direct binding of inhaled anesthetics to ligand-gated ion channels. This hypothesis is being studied using four-alpha-helix bundles as model systems for the transmembrane domains of the natural "receptor" proteins. This study concerns the role in anesthetic binding played by aromatic side chains in the binding cavity of a four-alpha-helix bundle designed to assume a Rop-like fold. Specifically, the effect of the substitution W15Y on bundle structure, stability, and anesthetic binding energetics was investigated. No appreciable effect of substituting W for Y on the secondary structure or the thermodynamic stability of the four-alpha-helix bundle was identified. However, the substitution W15Y resulted in about 6- and 3-fold decreases in halothane and chloroform binding affinities, respectively. This effect may reflect weaker dipole-aromatic quadrupole interactions between the aromatic side chain and the anesthetic in the tyrosine-containing species, which possesses the smaller aromatic ring system. For these anesthetic binding proteins, this class of interaction occurs when the permanent nonspherical distribution of electrons in the aromatic ring systems interact with the weakly acidic CH group of the anesthetics.     

Molecular dynamics simulation of a synthetic four-alpha-helix bundle that binds the anesthetic halothane.

The structural features of binding sites for volatile anesthetics are examined by performing a molecular dynamics simulation study of the synthetic four-alpha-helix bundles (Aalpha2)2, which are formed by association of two 62-residue di-alpha-helical peptides. The peptide bundle (Aalpha2)2 was designed by Johansson et al. [Biochemistry 37 (1998) 1421-1429] and was shown experimentally to have a high affinity for the binding of the anesthetic halothane (CF3CBrCIH) in a hydrophobic cavity. Since (Aalpha2)2 can exhibit either the anti or syn topologies, the two distinct bundles are simulated both in the presence and in the absence of halothane. Nanosecond length molecular dynamics trajectories were generated for each system at room temperature (T = 298 K). The structural and dynamic effects of the inclusion of halothane are compared, illustrating that the structures are stable over the course of the simulation; that the (Aalpha2)2 bundles have suitable pockets that can accommodate halothane; that the halothane remains in the designed hydrophobic cavity in close proximity to the Trp residues with a preferred orientation; and that the dimensions of the peptide are perturbed by the inclusion of an anesthetic molecule.     

Effect of four-alpha-helix bundle cavity size on volatile anesthetic binding energetics

Currently, it is thought that inhalational anesthetics cause anesthesia by binding to ligand-gated ion channels. This is being investigated using four-alpha-helix bundles, small water-soluble analogues of the transmembrane domains of the "natural" receptor proteins. The study presented here specifically investigates how multiple alanine-to-valine substitutions (which each decrease the volume of the internal binding cavity by 38 A(3)) affect structure, stability, and anesthetic binding affinity of the four-alpha-helix bundles. Structure remains essentially unchanged when up to four alanine residues are changed to valine. However, stability increases as the number of these substitutions is increased. Anesthetic binding affinities are also affected. Halothane binds to the four-alpha-helix bundle variants with 0, 1, and 2 substitutions with equivalent affinities but binds to the variants with 3 and 4 more tightly. The same order of binding affinities was observed for chloroform, although for a particular variant, chloroform was bound less tightly. The observed differences in binding affinities may be explained in terms of a modulation of van der Waals and hydrophobic interactions between ligand and receptor. These, in turn, could result from increased four-alpha-helix bundle binding cavity hydrophobicity, a decrease in cavity size, or improved ligand/receptor shape complementarity.     

Design and biophysical characterization of a monomeric four-alpha-helix bundle protein Aα4 with affinity for the volatile anesthetic halothane

A monomeric four-α-helix bundle protein Aα₄ was designed as a step towards investigating the interaction of volatile general anesthetics with their putative membrane protein targets. The alpha helices, connected by glycine loops, have the sequence A, B, B′, A′. The DNA sequence was designed to make the helices with the same amino acid sequences (helix A and A′, B and B′, respectively) as different as possible, while using codons which are favorable for expression in E. coli. The protein was bacterially expressed and purified to homogeneity using reversed-phase HPLC. Protein identity was verified using MALDI–TOF mass spectrometry. Far-UV circular dichroism spectroscopy confirmed the predominantly alpha-helical nature of the protein Aα₄. Guanidinium chloride induced denaturation showed that the monomeric four-α-helix bundle protein Aα₄ is considerably more stable compared to the dimeric di-α-helical protein (Aα₂-L38M)₂. The sigmoidal character of the unfolding reaction is conserved while the sharpness of the transition is increased 1.8-fold. The monomeric four-α-helix bundle protein Aα₄ bound halothane with a dissociation constant (K_d) of 0.93 ± 0.02 mM, as shown by both tryptophan fluorescence quenching and isothermal titration calorimetry. This monomeric four-α-helix bundle protein can now be used as a scaffold to incorporate natural central nervous system membrane protein sequences in order to examine general anesthetic interactions with putative targets in detail.     

Efficient generation of feasible pathways for protein conformational transitions

We develop a computationally efficient method to simulate the transition of a protein between two conformations. Our method is based on a coarse-grained elastic network model in which distances between spatially proximal amino acids are interpolated between the values specified by the two end conformations. The computational speed of this method depends strongly on the choice of cutoff distance used to define interactions as measured by the density of entries of the constant linking/contact matrix. To circumvent this problem we introduce the concept of using a cutoff based on a maximum number of nearest neighbors. This generates linking matrices that are both sparse and uniform, hence allowing for efficient computations that are independent of the arbitrariness of cutoff distance choices. Simulation results demonstrate that the method developed here reliably generates feasible intermediate conformations, because our method observes steric constraints and produces monotonic changes in virtual bond and torsion angles. Applications are readily made to large proteins, and we demonstrate our method on lactate dehydrogenase, citrate synthase, and lactoferrin. We also illustrate how this framework can be used to complement experimental techniques that partially observe protein motions.     

Peroxide-induced conformational transitions of peanut storage protein (arachin)

Several effects of peroxides on protein have been described in the literature. Some of the effects have been ascribed to alterations in protein conformation but this has never been examined directly. This paper described responses of peanut storage globulin (arachin) to rancid oil and hydrogen peroxide as measured by electrophoretic mobility, antigenicity and circular dichroism of the globulin. Rancid oil and 3% hydrogen peroxide had little effect on these properties but 30% hydrogen peroxide increased the mobility, abolished the antigenicity and reduced the ordered structural modes of the protein. The results indicated that changes in protein conformation do not occur after contact with organoleptically rancid oil or dilute peroxides but do occur with concentrated peroxides.     

Disorder-to-order conformational transitions in protein structure and its relationship to disease

Function in proteins largely depends on the acquisition of specific structures through folding at physiological time scales. Under both equilibrium and non-equilibrium states, proteins develop partially structured molecules that being intermediates in the process, usually resemble the structure of the fully folded protein. These intermediates, known as molten globules, present the faculty of adopting a large variety of conformations mainly supported by changes in their side chains. Taking into account that the mechanism to obtain a fully packed structure is considered more difficult energetically than forming partially “disordered” folding intermediates, evolution might have conferred upon an important number of proteins the capability to first partially fold and—depending on the presence of specific partner ligands—switch on disorder-to-order transitions to adopt a highly ordered well-folded state and reach the lowest energy conformation possible. Disorder in this context can represent segments of proteins or complete proteins that might exist in the native state. Moreover, because this type of disorder-to-order transition in proteins has been found to be reversible, it has been frequently associated with important signaling events in the cell. Due to the central role of this phenomenon in cell biology, protein misfolding and aberrant disorder-to-order transitions have been at present associated with an important number of diseases.     

Relationships between sequence and structure for the four-alpha-helix bundle tertiary motif in proteins.

The sequences of four-alpha-helical bundle proteins are characterized by a pattern of hydrophilic and hydrophobic amino acids which is repeated every seven residues. At each position of the heptad repeat there are specific constraints on the amino acid properties which result from the topology of the tertiary motif. These constraints give rise to patterns of amino acid distribution which are distinct from those of other proteins. The distributions in each of the heptad positions have been determined by a statistical analysis of structural and sequence data derived from seven families of aligned protein sequences. The constitution of each position is dominated by a very small number of different amino acids, with the core positions consisting overwhelmingly of Leu and Ala. The positional preferences of the individual amino acids can be generally interpreted in terms of residue properties and topological constraints. The potential for four-alpha-helix bundle folding is reflected primarily in the pattern of residue occurrence in the heptad and not in the overall amino acid composition of the protein. Possible applications of this analysis in structure predictions, sequence alignments and in the rational design and engineering of four-alpha-helical bundle proteins are discussed.     

Predicting order of conformational changes during protein conformational transitions using an interpolated elastic network model

The decryption of sequence of structural events during protein conformational transitions is essential to a detailed understanding of molecular functions ofvarious biological nanomachines. Coarse‐grained models have proven useful by allowing highly efficient simulations of protein conformational dynamics. By combining two coarse‐grained elastic network models constructed based on the beginning and end conformations of a transition, we have developed an interpolated elastic network model to generate a transition pathway between the two protein conformations. For validation, we have predicted the order of local and global conformational changes during key ATP‐driven transitions in three important biological nanomachines (myosin, F₁ ATPase and chaperonin GroEL). We have found that the local conformational change associated with the closing of active site precedes the global conformational change leading to mechanical motions. Our finding is in good agreement with the distribution of intermediate experimental structures, and it supports the importance of local motions at active site to drive or gate various conformational transitions underlying the workings of a diverse range of biological nanomachines. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The role of the 132-160 region in prion protein conformational transitions

The native conformation of host-encoded cellular prion protein (PrP(C)) is metastable. As a result of a post-translational event, PrP(C) can convert to the scrapie form (PrP(Sc)), which emerges as the essential constituent of infectious prions. Despite thorough research, the mechanism underlying this conformational transition remains unknown. However, several studies have highlighted the importance of the N-terminal region spanning residues 90-154 in PrP folding. In order to understand why PrP folds into two different conformational states exhibiting distinct secondary and tertiary structure, and to gain insight into the involvement of this particular region in PrP transconformation, we studied the pressure-induced unfolding/ refolding of recombinant Syrian hamster PrP expanding from residues 90-231, and compared it with heat unfolding. By using two intrinsic fluorescent variants of this protein (Y150W and F141W), conformational changes confined to the 132-160 segment were monitored. Multiple conformational states of the Trp variants, characterized by their spectroscopic properties (fluorescence and UV absorbance in the fourth derivative mode), were achieved by tuning the experimental conditions of pressure and temperature. Further insight into unexplored conformational states of the prion protein, likely to mimic the in vivo structural change, was obtained from pressure-assisted cold unfolding. Furthermore, salt-induced conformational changes suggested a structural stabilizing role of Tyr150 and Phe141 residues, slowing down the conversion to a beta-sheet form.