Positions Of Charged Residues Research Articles

Characterizing the Translocation of Charged Peptide Loops across Lipid Bilayers with Molecular Dynamics Simulations

The hydrophobic core of the cell membrane is considered largely impermeable to charged molecules because of the large free energy barrier and corresponding long timescale (∼hours) for charge translocation. Contradicting this view, a variety of water-soluble peptides are known to translocate charged groups across the cell membrane on a surprisingly rapid timescale, on the order of few seconds at least for some peptides. In this work, we study the interconversion of a peptide with charged flanking loops between a surface-adsorbed and membrane-embedded state, which requires the translocation (or “flipping”) of charged loops across the bilayer. We utilize all-atom Temperature Accelerated Molecular Dynamics (TAMD) simulations to predict the likelihood of loop flipping without predefining a specific translocation pathway. We show that membrane-exposed charged residues accelerate the flipping of charged peptide loops by stabilizing intramembrane water defects. We further demonstrate that this computational approach can identify multiple flipping pathways without specifying them a priori. The position of charged residues in the transmembrane helix also affects the flipping pathways and the apparent flipping free energy barrier. These detailed molecular-level insights of peptide translocation pathways may be valuable for designing small cationic peptides for applications in gene and drug delivery. Moreover, the methodology and findings discussed here can generalize to more complex behaviors, such as the large-scale conformational rearrangements of integral membrane proteins.

Effects of Charged Residues Inserted above R1 in S4-Based Voltage Sensors

Membrane proteins containing S4-based voltage sensors domains (VSD) respond to changes in the membrane potential by transferring electrically charged side chains (gating charges), mainly located in the S4 segment, across the membrane electric field. Yet, in the archetypical Shaker Kv channel, not all positive S4 residues contribute to the gating charge. Moreover, the number and positions of charged residues near the external side of S4 is poorly conserved amongst VSDs, suggesting that some S4 charged residues may have a regulatory role. We investigated this hypothesis by individually inserting Arg residues at the top of the S4 segment of the Shaker channel, from positions 356 to 361, and determining the impact of the mutations on the channel's gating currents. Our results show a non-ambiguous periodicity of the phenotypes that correlate with the periodicity of the S4 alpha-helical structure: Arg inserted on the same side of the gating charge positively-shifted the charge vs. voltage (Q-V) curve, while Arg inserted on the opposite side negatively-shifted the Q-V curve. We propose two distinct mechanisms that account for these observations. Charged side chains pointing towards the gating charge pathway exert a local bias in the electric field sensed by the innermost native Arg. However, charged side chains located on the opposite side of the gating charge pathway tend to favor more depolarized conformations of the S4 helix to avoid being in proximity to the hydrophobic environment of the membrane lipids. We propose that naturally-occurring charged residues located at the top of S4 may help to finely tune the VSD biophysical properties. This mechanism is illustrated by a myasthenia-associated muscle sodium channel variant that introduced the V1442E mutation at the top of the S4 in domain IV. This work was supported by NIH grant GM030376.

Structural Similarity and Classification of Protein Interaction Interfaces

Interactions between proteins play a key role in many cellular processes. Studying protein-protein interactions that share similar interaction interfaces may shed light on their evolution and could be helpful in elucidating the mechanisms behind stability and dynamics of the protein complexes. When two complexes share structurally similar subunits, the similarity of the interaction interfaces can be found through a structural superposition of the subunits. However, an accurate detection of similarity between the protein complexes containing subunits of unrelated structure remains an open problem.Here, we present an alignment-free machine learning approach to measure interface similarity. The approach relies on the feature-based representation of protein interfaces and does not depend on the superposition of the interacting subunit pairs. Specifically, we develop an SVM classifier of similar and dissimilar interfaces and derive a feature-based interface similarity measure. Next, the similarity measure is applied to a set of 2,806×2,806 binary complex pairs to build a hierarchical classification of protein-protein interactions. Finally, we explore case studies of similar interfaces from each level of the hierarchy, considering cases when the subunits forming interactions are either homologous or structurally unrelated. The analysis has suggested that the positions of charged residues in the homologous interfaces are not necessarily conserved and may exhibit more complex conservation patterns.

Effect of the charge distribution along the “ferritin-like” pores of the proteins from the Dps family on the iron incorporation process

DNA-binding proteins from starved cells (Dps) differ in the number and position of charged residues along the "ferritin-like" pores that are used by iron to reach the ferroxidase center and the protein cavity. These differences are shown to affect significantly the electrostatic potential at the pores, which determines the extent of cooperativity in the iron uptake kinetics and thereby the mass distribution of the ferric hydroxide micelles inside the protein cavity. These conclusions are of biotechnological value in the preparation of protein-enclosed nanomaterials and are expected to apply also to ferritins. They were reached after characterization of the Dps from Listeria innocua, Helicobacter pylori, Thermosynechococcus elongatus, Escherichia coli, and Mycobacterium smegmatis. The characterization comprised the calculation of the electrostatic potential at the pores, determination of the iron uptake kinetics in the presence of molecular oxygen or hydrogen peroxide, and analysis of the proteins by means of the sedimentation velocity after iron incorporation.

Deposition of apatite in mineralizing vertebrate extracellular matrices: A model of possible nucleation sites on type I collagen

The positions of charged residues in the primary sequence of amino acids comprising the molecular model of type I collagen, the major extracellular protein found in vertebrate tissues, have been earlier characterized by Chapman and Hardcastle [Chapman, J.A., and Hardcastle, R.A. (1974). The staining pattern of collagen fibrils. II. A comparison with patterns computer-generated from the amino acid sequence. Connect. Tissue Res. 2:151–159]. When the sequence of residues is packed in the quarter-staggered arrangement described originally by Hodge and Petruska [Hodge, A.J., and Petruska, J.A. (1963). Recent studies with the electron microscope on ordered aggregates of the tropocollagen macromolecule. In Aspects of Protein Structure, G.N. Ramachandran (ed.) pp. 289–300. New York: Academic Press] in two dimensions and in the quasi-hexagonal model of microfibrillar assembly and molecular packing structure in three dimensions detailed recently by Orgel et al. (Orgel, J.P.R.O., Miller, A., Irving, T.C., Fischetti, R.F., Hammersley, A.P., and Wess, T.J. (2001). The in situ supermolecular structure of type I collagen. Structure 9:1061–1069; Orgel, J.P.R.O., Irving, T.C., Miller, A., and Wess, T.J. (2006). Microfibrillar structure of type I collagen in situ. Proc. Natl. Acad. Sci. U.S.A. 103: 9001–9005], the common sites of charged amino acids, specifically glutamic and aspartic acid, lysine and arginine, and hydroxylysine and histidine, of type I collagen have been examined in the present study and their locations determined in relation to one another. The respective positions of these amino acid residues are notable in several features in two dimensions within a single collagen triple helix as well as in adjacent helices. There are, first, numerous sites in which the same amino acid is adjacent in each of the three collagen helices. Second, many sites exist in which two of the same amino acids and one of the same charge are adjacent in the three helices. Third, the same two or three glutamic and/or aspartic amino acids are found in close proximity to amino acids with their counterparts, aspartic and glutamic acid, respectively. Fourth, several sites occur in which the same two or three amino acids of one charge are present in close proximity to the same two or three amino acids of opposite charge (glutamic acid and lysine or arginine residues or aspartic acid and lysine or arginine residues). Fifth, there are several sites where hydroxylysine contributes charged groups in place of one of the three lysine or arginine residues common in adjacent collagen helices. The strikingly repetitive and close nature of these specific charged groups in two dimensions is even more apparent when the molecular packing structure is investigated in three dimensions. In this instance, the most recent model of Orgel et al. [Orgel, J.P.R.O., Irving, T.C., Miller, A., and Wess, T.J. (2006). Microfibrillar structure of type I collagen in situ. Proc. Natl. Acad. Sci. U.S.A. 103: 9001–9005] has been correlated for the first time with the model of Landis et al. [Landis, W.J., Song, M.J., Leith, A., McEwen, L., and McEwen, B. (1993). Mineral and organic matrix interaction in normally calcifying tendon visualized in three dimensions by high voltage electron microscopic tomography and graphic image reconstruction. J. Struct. Biol. 110: 39–54] showing channels traversing molecular arrays of collagen. Here, many of the charged amino acid sites correspond to the known type I collagen hole zones defined by Hodge and Petruska [Hodge, A.J., and Petruska, J.A. (1963). Recent studies with the electron microscope on ordered aggregates of the tropocollagen macromolecule. In Aspects of Protein Structure, G.N. Ramachandran (ed.) pp. 289–300. New York: Academic Press]. As such, these residues present the locations highly likely to bind Ca2+ and ions in stereochemical configurations that could serve directly as nucleation centers for the subsequent growth and development of apatite crystals representing initial events in vertebrate mineralization. Based on these results, type I collagen appears to provide a molecular framework for direct formation of apatite without the necessary intervention or mediation of other molecules in extracellular matrices of vertebrate calcifying tissues.

Disruption Mechanism in the Helix of SPF Peptide by Interchanging E5 and K10 Residues: Inference from Molecular Dynamics Study

Seminalplasmin (SPLN) is a 47-residue peptide (SDEKASPDKHHRFSLSRYAKLANR LANPKLLETFLSKWIGDRGNRSV) from bovine seminal plasma. It has broad spectrum antimicrobial activity, without any hemolytic activity. The 28–40 segment of SPLN with the sequence PKLLETFLSKWIG, designated as SPF, is the most hydrophobic stretch of SPLN and primarily responsible for the membrane-perturbing activity of SPLN. It was reported that SPF has a helical structure and the interchange of E5 and K10 residues disrupted the helical structure. The present paper reports a possible mechanism of disruption of the helical structure of SPF peptide during the interchange of E5 and K10 residues. The result is based on simulated annealing and molecular dynamics simulation studies on SPF and its four analogues with K10E, K10D, E5K, and E5K & K10E substitutions. It showed that K10 residue has a critical role in maintaining the highest helical content and the positions of charged residues are also very important for maintaining the helical structure of the SPF peptide. Formation of some new long-range hydrogen bonds and the rupture of some shortrange hydrogen bonds involving the tenth residue led to the disruption of helical structure of SPF peptide when E5 and K10 residues are interchanged.

Role of Charged Residues in the S1–S4 Voltage Sensor of BK Channels

The activation of large conductance Ca2+-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K+ (KV) channels. Yet BK and KV channels share many conserved charged residues in transmembrane segments S1–S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (Po) and IK kinetics (τ(IK)) over an extended voltage range in 0–50 μM [Ca2+]i. mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of PO. The voltage dependence of PO and τ(IK) at extreme negative potentials was also reduced, implying that the closed–open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and KV channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to KV channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1–S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3–7 kcal mol−1, indicating a strong contribution of non–voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.

The Amino Acid Sequence in Fibrin Responsible for High Affinity Thrombin Binding

Human fibrin has a low affinity thrombin binding site in its E domain and a high affinity binding site in the carboxy-terminal region of its variant gamma' chain (gamma'408-427). Comparison of the gamma' amino acid sequence (VRPEHPAETEYDSLYPEDDL) with other protein sequences known to bind to thrombin exosites such as those in GPIbalpha, the platelet thrombin receptor, thrombomodulin, and hirudin suggests no homology or consensus sequences, but Glu and Asp enrichment are common to all. Tyrosine sulfation in these sequences enhances thrombin exosite binding, but this has not been uniformly investigated. The fibrinogen gamma' chain mass determined by electrospray ionization mass spectrometry, was 50,549 Da, a value 151 Da greater than predicted from its amino acid/carbohydrate sequence. Since each sulfate group increases mass by 80 Da, this indicates that both tyrosines at 418 and 422 are sulfated. A series of overlapping gamma' peptides was prepared for evaluation of their inhibition of 125I-labeled PPACK-thrombin binding to fibrin. gamma'414-427 was as effective an inhibitor as gamma'408-427 and its binding affinity was dependent on all carboxy-terminal residues. Mono Tyr-sulfated peptides were prepared by substituting non-sulfatable Phe for Tyr at gamma'418 or 422. Sulfation at either Tyr residue increased binding competition compared with non-sulfated peptides, but was less effective than doubly sulfated peptides, which had 4 to 8-fold greater affinity. The reverse gamma' peptide or the forward sequence with repositioned Tyr residues did not compete well for thrombin binding, indicating that the positions of charged residues are important for thrombin binding affinity.

Molecular determinants of electrical rectification of single channel conductance in gap junctions formed by connexins 26 and 32.

The fully open state of heterotypic gap junction channels formed by pairing cells expressing connexin 32 (Cx32) with those expressing connexin 26 (Cx26) rectifies in a way that cannot be predicted from the current-voltage (I-V) relation of either homotypic channel. Using a molecular genetic analysis, we demonstrate that charged amino acids positioned in the amino terminus (M1 and D2) and first extracellular loop (E42) are major determinants of the current-voltage relation of the fully open state of homotypic and heterotypic channels formed by Cx26 and Cx32. The observed I-V relations of wild-type and mutant channels were closely approximated by those obtained with the electrodiffusive model of Chen and Eisenberg (Chen, D., and R. Eisenberg. 1993. Biophys. J. 64:1405-1421), which solves the Poisson-Nernst-Plank equations in one dimension using charge distribution models inferred from the molecular analyses. The rectification of the Cx32/Cx26 heterotypic channel results from the asymmetry in the number and position of charged residues. The model required the incorporation of a partial charge located near the channel surface to approximate the linear I-V relation observed for the Cx32*Cx26E1 homotypic channel. The best candidate amino acid providing this partial charge is the conserved tryptophan residue (W3). Incorporation of the partial charge of residue W3 and the negative charge of the Cx32E41 residue into the charge profile used in the Poisson-Nernst-Plank model of homotypic Cx32 and heterotypic Cx26/Cx32 channels resulted in I-V relations that closely resembled the observed I-V relations of these channels. We further demonstrate that some channel substates rectify. We suggest that the conformational changes associated with transjunctional voltage (V(j))-dependent gating to these substates involves a narrowing of the cytoplasmic entry of the channel that increases the electrostatic effect of charges in the amino terminus. The rectification that is observed in the Cx32/Cx26 heterotypic channel is similar although less steep than that reported for some rectifying electrical synapses. We propose that a similar electrostatic mechanism, which results in rectification through the open and substates of heterotypic channels, is sufficient to explain the properties of steeply rectifying electrical synapses.

Studies of synthetic peptide analogs of the amphipathic helix. Effect of charge distribution, hydrophobicity, and secondary structure on lipid association and lecithin:cholesterol acyltransferase activation.

Four peptides capable of forming an amphipathic alpha-helix have been synthesized and their conformational and lipid-binding properties studied. These peptides have been designed to vary the alpha-helix-forming potential as well as the charge distribution of the model peptide. The resulting peptide analogs and their complexes with dimyristoyl phosphatidylcholine were studied by using right angle light scattering, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, circular dichroism, intrinsic tryptophan fluorescence, and differential scanning calorimetry techniques. The four analogs, [Glu4,9, Leu11,17] (reverse-18A, [Glu4,9, Leu5,11,17] reverse-18A, [Glu1,8, Leu11,17] 18A, and [Glu1,8, Leu5,11,17] 18A were derived from a model amphipathic peptide Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) whose lipid-associating properties strongly mimic apolipoprotein A-I or derived from Lys-Trp-Leu-Asp-Ala-Phe-Tyr-Lys-Asp-Val-Ala-Lys-Glu-Leu-Glu-Lys-Ala-Phe (reverse-18A), a peptide with little affinity for lipid and having a reversed charge distribution compared to the 18A peptide. We have shown that by substituting glutamic acid and leucine for aspartic acid and alanine, respectively, in a weak lipid-associating amphipathic helix peptide, the lipid-associating ability can be increased. Thus, peptides with both kinds of charge distribution can associate with the lipid. The ability of the peptide to disrupt phospholipid bilayers, however, is higher for 18A analogs compared to the reverse-18A analogs even after increasing the helix-forming potential and hydrophobicity. In addition to forming smaller lipoprotein particles, the modified 18A analogs were much superior to the modified reverse-18A analogs in their ability to activate the enzyme lecithin:cholesterol acyltransferase. This demonstrates that the positions of charged residues in the amphipathic helix play an important role in lecithin:cholesterol acyltransferase activation.