Hydrophobic Moment Research Articles

PAGAL - Properties and corresponding graphics of alpha helical structures in proteins

Alpha helices (AH) are peptide fragments characterized by regular patterns of hydrogen bonding between the carbonyl oxygen and amino nitrogen of residues regularly spaced in sequence, resulting in spiral conformations. Their preponderance in protein structures underlines their importance. Interestingly, AHs are present in most anti-microbial peptides, although they might remain in random-coil conformations depending on the solvent dielectric. For example, the cecropin component of the chimeric anti-microbial protein designed previously by our group comprises of two AHs linked by a short stretch of random coil. These anti-microbial peptides are often amphipathic (quantified by a hydrophobic moment), aligning hydrophobic residues on one surface and charged residues on the others. In the current work, we reproduce previously described computational methods to compute the hydrophobic moment of AHs - and provide open access to the source code (PAGAL). We simultaneously generated input files for TikZ (a package for creating high resolution graphics programmatically) to obtain the Edmundson wheel and showing the direction and magnitude of the hydrophobic moment, and Pymol scripts to generate color coded protein surfaces. Additionally, we have observed an empirical structural property of AHs: the distance between the Cα atoms of the ith and (i+4)th residue is equal to the distance between the carbonyl oxygens of the ith and (i+4)th residue. We validated this using 100 non-homologous high resolution structures from the PISCES database. The source code and manual is available at http://github.com/sanchak/pagal and on http://dx.doi.org/10.5281/zenodo.11136.

PAGAL - Properties and corresponding graphics of alpha helical structures in proteins

Alpha helices (AH) are peptide fragments characterized by regular patterns of hydrogen bonding between the carbonyl oxygen and amino nitrogen of residues regularly spaced in sequence, resulting in spiral conformations. Their preponderance in protein structures underlines their importance. Interestingly, they are invariably present in all anti-microbial peptides. For example, the cecropin component of the chimeric anti-microbial protein designed previously by our group comprises of two AHs linked by a short stretch of random coil. These anti-microbial peptides are often amphipathic (quantified by a hydrophobic moment), aligning hydrophobic residues on one surface and charged residues on the others. In the current work, we reproduce previously described computational methods to compute the hydrophobic moment of AHs - and provide open access to the source code (PAGAL). We simultaneously generated input files for TikZ (a package for creating high resolution graphics programmatically) to obtain the Edmundson wheel and showing the direction and magnitude of the hydrophobic moment, and Pymol scripts to generate color coded protein surfaces. Additionally, we have observed an empirical structural property of AHs: the distance between the Cα atoms of the ith and (i+4)th residue is equal to the distance between the carbonyl oxygens of the ith and (i+4)th residue. We validated this using 100 non-homologous high resolution structures from the PISCES database. The source code and manual is available at http://github.com/sanchak/pagal and on http://dx.doi.org/10.5281/zenodo.11136.

3D Hydrophobic Moment Vectors as a Tool to Characterize the Surface Polarity of Amphiphilic Peptides

The interaction of membranes with peptides and proteins is largely determined by their amphiphilic character. Hydrophobic moments of helical segments are commonly derived from their two-dimensional helical wheel projections, and the same is true for β-sheets. However, to the best of our knowledge, there exists no method to describe structures in three dimensions or molecules with irregular shape. Here, we define the hydrophobic moment of a molecule as a vector in three dimensions by evaluating the surface distribution of all hydrophilic and lipophilic regions over any given shape. The electrostatic potential on the molecular surface is calculated based on the atomic point charges. The resulting hydrophobic moment vector is specific for the instantaneous conformation, and it takes into account all structural characteristics of the molecule, e.g., partial unfolding, bending, and side-chain torsion angles. Extended all-atom molecular dynamics simulations are then used to calculate the equilibrium hydrophobic moments for two antimicrobial peptides, gramicidin S and PGLa, under different conditions. We show that their effective hydrophobic moment vectors reflect the distribution of polar and nonpolar patches on the molecular surface and the calculated electrostatic surface potential. A comparison of simulations in solution and in lipid membranes shows how the peptides undergo internal conformational rearrangement upon binding to the bilayer surface. A good correlation with solid-state NMR data indicates that the hydrophobic moment vector can be used to predict the membrane binding geometry of peptides. This method is available as a web application on http://www.ibg.kit.edu/HM/.

In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors

Molecular docking of a library of all 8000 possible tripeptides to the active site of DPP-IV was used to determine their binding potential. A number of tripeptides were selected for experimental testing, however, there was no direct correlation between the Vina score and their in vitro DPP-IV inhibitory properties. While Trp-Trp-Trp, the peptide with the best docking score, was a moderate DPP-IV inhibitor (IC50 216μM), Lineweaver and Burk analysis revealed its action to be non-competitive. This suggested that it may not bind to the active site of DPP-IV as assumed in the docking prediction. Furthermore, there was no significant link between DPP-IV inhibition and the physicochemical properties of the peptides (molecular mass, hydrophobicity, hydrophobic moment (μH), isoelectric point (pI) and charge). LIGPLOTs indicated that competitive inhibitory peptides were predicted to have both hydrophobic and hydrogen bond interactions with the active site of DPP-IV. DPP-IV inhibitory peptides generally had a hydrophobic or aromatic amino acid at the N-terminus, preferentially a Trp for non-competitive inhibitors and a broader range of residues for competitive inhibitors (Ile, Leu, Val, Phe, Trp or Tyr). Two of the potent DPP-IV inhibitors, Ile-Pro-Ile and Trp-Pro (IC50 values of 3.5 and 44.2μM, respectively), were predicted to be gastrointestinally/intestinally stable. This work highlights the needs to test the assumptions (i.e. competitive binding) of any integrated strategy of computational and experimental screening, in optimizing screening. Future strategies targeting allosteric mechanisms may need to rely more on structure–activity relationship modeling, rather than on docking, in computationally selecting peptides for screening.

TSpred: a web server for the rational design of temperature-sensitive mutants.

Temperature sensitive (Ts) mutants of proteins provide experimentalists with a powerful and reversible way of conditionally expressing genes. The technique has been widely used in determining the role of gene and gene products in several cellular processes. Traditionally, Ts mutants are generated by random mutagenesis and then selected though laborious large-scale screening. Our web server, TSpred (http://mspc.bii.a-star.edu.sg/TSpred/), now enables users to rationally design Ts mutants for their proteins of interest. TSpred uses hydrophobicity and hydrophobic moment, deduced from primary sequence and residue depth, inferred from 3D structures to predict/identify buried hydrophobic residues. Mutating these residues leads to the creation of Ts mutants. Our method has been experimentally validated in 36 positions in six different proteins. It is an attractive proposition for Ts mutant engineering as it proposes a small number of mutations and with high precision. The accompanying web server is simple and intuitive to use and can handle proteins and protein complexes of different sizes.

Prediction of linear cationic antimicrobial peptides based on characteristics responsible for their interaction with the membranes.

Most available antimicrobial peptides (AMP) prediction methods use common approach for different classes of AMP. Contrary to available approaches, we suggest that a strategy of prediction should be based on the fact that there are several kinds of AMP that vary in mechanisms of action, structure, mode of interaction with membrane, etc. According to our suggestion for each kind of AMP, a particular approach has to be developed in order to get high efficacy. Consequently, in this paper, a particular but the biggest class of AMP, linear cationic antimicrobial peptides (LCAP), has been considered and a newly developed simple method of LCAP prediction described. The aim of this study is the development of a simple method of discrimination of AMP from non-AMP, the efficiency of which will be determined by efficiencies of selected descriptors only and comparison the results of the discrimination procedure with the results obtained by more complicated discriminative methods. As descriptors the physicochemical characteristics responsible for capability of the peptide to interact with an anionic membrane were considered. The following characteristics such as hydrophobicity, amphiphaticity, location of the peptide in relation to membrane, charge density, propensities to disordered structure and aggregation were studied. On the basis of these characteristics, a new simple algorithm of prediction is developed and evaluation of efficacies of the characteristics as descriptors performed. The results show that three descriptors, hydrophobic moment, charge density and location of the peptide along the membranes, can be used as discriminators of LCAPs. For the training set, our method gives the same level of accuracy as more complicated machine learning approaches offered as CAMP database service tools. For the test set accuracy obtained by our method gives even higher value than the one obtained by CAMP prediction tools. The AMP prediction tool based on the considered method is available at http://www.biomedicine.org.ge/dbaasp/.

The Role of Protein Hydrophobicity in Conformation Change and Self-Assembly into Large Amyloid Fibers

It has been found that a short hydrophobic "template" peptide and a larger α-helical "adder" protein cooperatively self-assemble into micrometer sized amyloid fibers. Here, a common template of trypsin hydrolyzed gliadin is combined with six adder proteins (α-casein, α-lactalbumin, amylase, hemoglobin, insulin, and myoglobin) to determine what properties of the adder protein drive amyloid self-assembly. Utilizing Fourier Transform-Infrared (FT-IR) spectroscopy, the Amide I absorbance reveals that the observed decrease in α-helix with time is approximately equal to the increase in high strand density β-sheet, which is indicative of amyloid formation. The results show that the hydrophobic moment is a good predictor of conformation change but the fraction of aliphatic amino acids within the α-helices is a better predictor. Upon drying, the protein mixtures form large amyloid fibers. The fiber twist is dependent on the aliphatic index and molecular weight of the adder protein. Here we demonstrate that it is possible to predict the propensity of an adder protein to unfold into an amyloid structure and to predict the fiber morphology, both from adder protein molecular features, which can be applied to the pragmatic engineering of large amyloid fibers.

Alzheimer’s Aβ10–40 Peptide Binds and Penetrates DMPC Bilayer: An Isobaric–Isothermal Replica Exchange Molecular Dynamics Study

Using all-atom explicit solvent model and isobaric-isothermal replica exchange molecular dynamics, we studied binding of Aβ10-40 monomers to zwitterionic DMPC bilayer. Our simulations suggest three main conclusions. First, binding of Aβ10-40 monomer to the DMPC bilayer causes dramatic structural transition in the peptide resulting in the formation of stable helical structure in the C-terminal. In addition, binding to the lipid bilayer induces the formation of intrapeptide Asp23-Lys28 salt bridge. We argue that the emergence of helix is the consequence of hidden helix propensity harbored in the Aβ10-40 C-terminal. This propensity is revealed by the lipids cross-bridging amino acids in helical conformations and by significant hydrophobic moment of the C-terminal. Second, the central hydrophobic cluster and, particularly, the C-terminal of Aβ10-40 not only govern binding to the bilayer but also penetrate into bilayer core. In contrast, the polar N-terminal and turn region form interactions mainly with the bilayer surface. Third, our simulations suggest that upon Aβ10-40 binding to the bilayer a highly heterogeneous local environment emerges along Aβ10-40 chain. The N-terminal is exposed to polar well-hydrated medium, whereas the C-terminal is largely shielded from water residing in mostly hydrophobic environment. The implication of our results is that Aβ aggregation mediated by zwitterionic lipid bilayer is likely to be different from that in bulk water.

High-resolution structures and orientations of antimicrobial peptides piscidin 1 and piscidin 3 in fluid bilayers reveal tilting, kinking, and bilayer immersion.

While antimicrobial peptides (AMPs) have been widely investigated as potential therapeutics, high-resolution structures obtained under biologically relevant conditions are lacking. Here, the high-resolution structures of the homologous 22-residue long AMPs piscidin 1 (p1) and piscidin 3 (p3) are determined in fluid-phase 3:1 phosphatidylcholine/phosphatidylglycerol (PC/PG) and 1:1 phosphatidylethanolamine/phosphatidylglycerol (PE/PG) bilayers to identify molecular features important for membrane destabilization in bacterial cell membrane mimics. Structural refinement of 1H–15N dipolar couplings and 15N chemical shifts measured by oriented sample solid-state NMR and all-atom molecular dynamics (MD) simulations provide structural and orientational information of high precision and accuracy about these interfacially bound α-helical peptides. The tilt of the helical axis, τ, is between 83° and 93° with respect to the bilayer normal for all systems and analysis methods. The average azimuthal rotation, ρ, is 235°, which results in burial of hydrophobic residues in the bilayer. The refined NMR and MD structures reveal a slight kink at G13 that delineates two helical segments characterized by a small difference in their τ angles (<10°) and significant difference in their ρ angles (∼25°). Remarkably, the kink, at the end of a G(X)4G motif highly conserved among members of the piscidin family, allows p1 and p3 to adopt ρ angles that maximize their hydrophobic moments. Two structural features differentiate the more potent p1 from p3: p1 has a larger ρ angle and less N-terminal fraying. The peptides have comparable depths of insertion in PC/PG, but p3 is 1.2 Å more deeply inserted than p1 in PE/PG. In contrast to the ideal α-helical structures typically assumed in mechanistic models of AMPs, p1 and p3 adopt disrupted α-helical backbones that correct for differences in the amphipathicity of their N- and C-ends, and their centers of mass lie ∼1.2–3.6 Å below the plane defined by the C2 atoms of the lipid acyl chains.

Peptides with the Same Composition, Hydrophobicity, and Hydrophobic Moment Bind to Phospholipid Bilayers with Different Affinities

We investigated the dependence of membrane binding on amino acid sequence for a series of amphipathic peptides derived from δ-lysin. δ-Lysin is a 26 amino acid, N-terminally formylated, hemolytic peptide that forms an amphipathic α-helix bound at membrane-water interfaces. A shortened peptide, lysette, was derived from δ-lysin by deletion of the four N-terminal amino acid residues. Five variants of lysette were synthesized by altering the amino acid sequence such that the overall hydrophobic moment remained essentially the same for all peptides. Peptide-lipid equilibrium dissociation constants and helicities of peptides bound to zwitterionic lipid vesicles were determined by stopped-flowfluorescence and circular dichroism. We found that binding to phosphatidylcholine bilayers was a function of the helicity of the bound peptide alone and independent of the a priori hydrophobic moment or the ability to form intramolecular salt bridges. Molecular dynamics (MD) simulations on two of the peptides suggest that sequence determines the insertion depth into the bilayer. However, deeper bilayer insertion observed in the MD simulations is not synonymous with slow peptide desorption from the bilayer, as determined experimentally. We also found a systematic deviation of the experimentally determined dissociation constant and that predicted by the Wimley-White interfacial hydrophobicity scale. The reason for the discrepancy remains unresolved but appears to correlate with a predominance of isoleucine over leucine residues in the lysette family of peptides.

Antimicrobial Peptides Piscidin 1 and Piscidin 3 Kink at a Central Glycine to Maximize their Hydrophobic Moments

Piscidin 1 (p1) and the less active piscidin 3 (p3) are α-helical, amphipathic, and cationic antimicrobial peptides. They adsorb onto the outer membrane of pathogenic species and induce leakage beyond a threshold peptide concentration. While different mechanisms of membrane disruption have been proposed, an atomic-level description of the events leading to cell death is lacking. Elucidating these mechanisms is critical in the design of new antibiotics. Here, the structures, orientations, and positions of p1 and p3 in two different bacterial membrane mimics (3:1 DMPC/DMPG and 1:1 POPE/POPG) are studied by solid-state NMR spectroscopy, all-atom molecular dynamics (MD) simulations, and neutron diffraction to better understand molecular determinants that contribute to antimicrobial activity. The tilts of the helical axis, τ, are perpendicular to the bilayer normal (83-93°) and the average azimuthal rotation, ρ, corresponds to burial of hydrophobic residues in the bilayer. The refined NMR and MD structures reveal a slight kink at Gly13 of the α-helix. This kink is characterized by a small difference in τ (<10°) and significant difference in ρ (∼20°) and maximizes the hydrophobic moment for the α-helix. The depths of insertion are similar in DMPC/DMPG. In contrast, p1 and p3 are 1.0 and 3.0 A less inserted in POPE/POPG. Insertion of the peptides corresponds to greater thinning of the bilayer and a decrease in the lipid order parameter. Deformation of the DMPC/DMPG bilayer is characterized by uniform membrane thinning around the peptide, while deformation in the POPE/POPG bilayer is characterized by localized thinning near the N-terminus of the peptide. The increase in thinning in DMPC/DMPG relates to a greater activity than in POPE/POPG in dye-leakage assays and may be a precursor to a more disruptive state.

Peptide:Lipid Ratio and Membrane Surface Charge Modulate the Mechanism of Action of the Antimicrobial Peptide BP100

We present a comprehensive study of the conformational and functional properties of BP100 (H-KKLFKKILKYL-NH2) upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of zwitterionic PC and negatively charged PG. Theoretical prediction and calculation of hydrophobic moment indicate that BP100 is a surface-seeking amphipathic helix. Accordingly, CD spectra showed that, upon binding to PG-containing LUVs, BP100 acquires α-helical conformation. NMR data showed that the helix spans residues 3-11. Moreover, CD spectra evinced peptide aggregation in the membrane and/or vesicle aggregation, as a function of peptide:lipid ratio and PG content. Dynamic light scattering confirmed vesicle aggregation under conditions of electroneutralization. BP100 decreased the absolute value of the zeta (ζ)potential of LUVs with lower PGcontents. For higher PG contents, the ζ potential remained initially constant, a decrease occurring when ca. 80% of the negatively charged lipids were neutralized. This effect was rationalized in terms of an ion exchange process. Higher peptide concentrations were required for leakage from LUVs aqueous inner compartment at high ionic strength. Moreover, while a gradual release took place at low peptide:lipid ratios, instantaneous lossoccurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs also evinced BP100-promoted disruption of negatively charged membranes. We propose that the mechanism of action of BP100 is a function of both peptide:lipid ratio and negatively charged lipid content. While the gradual release of inner contents is a consequence of membrane perturbation by a small number of peptide molecules (monomers or small aggregates), membrane disruption results from a sequence of events: peptide aggregation on the membrane surface, leading to lipid clustering, andgiving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.

Lipid exposure prediction enhances the inference of rotational angles of transmembrane helices

BackgroundSince membrane protein structures are challenging to crystallize, computational approaches are essential for elucidating the sequence-to-structure relationships. Structural modeling of membrane proteins requires a multidimensional approach, and one critical geometric parameter is the rotational angle of transmembrane helices. Rotational angles of transmembrane helices are characterized by their folded structures and could be inferred by the hydrophobic moment; however, the folding mechanism of membrane proteins is not yet fully understood. The rotational angle of a transmembrane helix is related to the exposed surface of a transmembrane helix, since lipid exposure gives the degree of accessibility of each residue in lipid environment. To the best of our knowledge, there have been few advances in investigating whether an environment descriptor of lipid exposure could infer a geometric parameter of rotational angle.ResultsHere, we present an analysis of the relationship between rotational angles and lipid exposure and a support-vector-machine method, called TMexpo, for predicting both structural features from sequences. First, we observed from the development set of 89 protein chains that the lipid exposure, i.e., the relative accessible surface area (rASA) of residues in the lipid environment, generated from high-resolution protein structures could infer the rotational angles with a mean absolute angular error (MAAE) of 46.32˚. More importantly, the predicted rASA from TMexpo achieved an MAAE of 51.05˚, which is better than 71.47˚ obtained by the best of the compared hydrophobicity scales. Lastly, TMexpo outperformed the compared methods in rASA prediction on the independent test set of 21 protein chains and achieved an overall Matthew’s correlation coefficient, accuracy, sensitivity, specificity, and precision of 0.51, 75.26%, 81.30%, 69.15%, and 72.73%, respectively. TMexpo is publicly available at http://bio-cluster.iis.sinica.edu.tw/TMexpo.ConclusionsTMexpo can better predict rASA and rotational angles than the compared methods. When rotational angles can be accurately predicted, free modeling of transmembrane protein structures in turn may benefit from a reduced complexity in ensembles with a significantly less number of packing arrangements. Furthermore, sequence-based prediction of both rotational angle and lipid exposure can provide essential information when high-resolution structures are unavailable and contribute to experimental design to elucidate transmembrane protein functions.

The helical hairpin structure of the influenza fusion peptide can be seen on a hydrophobic moment map

An assignment of the helical hairpin of the influenza fusion peptide has been made based on the hydrophobic moments, represented in a form of two-dimensional map. Such assignment holds for all serotypes, even for the cases of mutations altering the amino acid character. Similar results are obtained for the experimentally developed hydrophobicity scales, whose values reflect the transfer energies between aqueous and membrane environments. A distinct, however still structure-related hydrophobic map corresponds to a helical and contiguous HIV gp41 fp. The method may be used as a simple tool for sequence-based prediction of structures adopted by viral fusion peptides.

Flexibility is a mechanical determinant of antimicrobial activity for amphipathic cationic α-helical antimicrobial peptides

Antimicrobial peptides (AMPs) are recognized as the potential substitutions for common antibiotics. Flexibility has been demonstrated to be a dominant on antimicrobial activity of an AMP, similar to the structural parameters such as hydrophobicity and hydrophobic moment as well as positive charge. To better understand the effect of flexibility on antimicrobial activity, we herein examined seventy-eight peptides derived from nine different species. Defined as a weighted average of amino acid flexibility indices over whole residue chain of AMP, flexibility index was used to scale the peptide flexibility and indicated to be a reflection of mechanical properties such as tensile and flexural rigidities. The results demonstrated that flexibility index is relevant to but different from other structural properties, may enhance activity against Escherichia coli for stiff clustered peptides or reduce activity against E. coli for flexible clustered peptides, and its optimum occurs at about −0.5. This effect of flexibility on antimicrobial activity may be involved to the antimicrobial actions, such as stable peptide-bound leaflet formation and sequent stress concentration in target cell membrane, mechanically. The present results provide a new insight in understanding antimicrobial actions and may be useful in seeking for a new structure–activity relationship for cationic and amphipathic α-helical peptides.

International Society for Computational Biology Honors David Eisenberg with 2013 Accomplishment by a Senior Scientist Award

International Society for Computational Biology Honors David Eisenberg with 2013 Accomplishment by a Senior Scientist Award

Amino Acid Sequence and Membrane Binding for a Series of Closely related Amphipathic Peptides

We have investigated the dependence of peptide activity on the amino acid sequence for a series of synthetic peptides derived from δ-lysin. δ-Lysin is a 26 amino acid, N-terminally formylated, hemolytic peptide that forms an amphipathic α-helix bound at membrane-water interfaces. A shortened peptide, lysette, was derived from δ-lysin by deletion of the 4 N-terminal amino acid residues. Five variants of lysette were synthesized by altering the amino acid sequence under the constraint that the overall hydrophobic moment be essentially the same for all peptides. Peptide-lipid equilibrium dissociation constants and helicities of peptides bound to zwitterionic lipid vesicles were determined by stopped-flow fluorescence and circular dichroism. We then compared the thermodynamics of peptide binding calculated using the Wimley-White hydrophobicity scale with the experimentally determined free energy of binding. We found a systematic deviation of the experimentally determined dissociation constant and that predicted by the Wimley-White scale. Molecular dynamics simulations suggest two factors that account for the very favorable experimental binding free energy. (1), in all δ-lysin-derived peptides simulated, the initial bilayer contact is made with the polar peptide face, allowing charged residues to establish strong interactions with the bilayer headgroup region early on. (2), if the two aspartate residues contained in the lysette sequences are located at the C-terminus, they remain exposed to water and are, thus, effectively removed from the bilayer headgroup region.

In silico Based Structural Analysis of Some Piperidine Analogs as Farnesyltransferase Inhibitors

In silico based QSAR and pharmacophore analyses of a series of piperidine derivatives were performed in order to investigate the structural features of the derivatives responsible for FTase inhibitory activity. The results derived from the QSAR analysis show that the FTase inhibitory activity mediated by the vdW surface area features such as partial charge (PEOE_VSA and Q_VSA) and v_surf (hydrophobic integy moment) of the molecules. The positive contribution of the partial charge descriptors such as Q_VSA_FPNEG and PEOE_VSA-4 reveal that the fractional negative charge on the vdW surface of the molecules and the aqueous solubility (LogS) of the molecules are important for the FTase inhibitory activity. While the hydrophobic integy moment reveals that a clear separation between the hydrophobic and the hydrophilic region in the molecules is important with electrostatic groups (fractional negative charge) for better activity. The pharmacophore analyses of the piperidine derivatives also show that the aromatic, acceptor and donor groups on the molecule favorable for the FTase inhibitory activity.

Hydrophobic Moments, Shape, and Packing in Disordered Proteins

Disordered proteins play a significant role in many biological processes and provide an attractive target for biophysical studies under physiological conditions. Disordered proteins may be classified as (a) proteins with overall well-defined secondary structures, interspersed with regions of missing residues, or (b) natively unstructured proteins which lack definite secondary structure. The spatial profile of second order hydrophobic moment for disordered proteins depicts the distribution of hydrophobic residues from the interior to the surface of the protein and indicates the lack of a well-formed hydrophobic core unlike that of the globular proteins. This trend is independent of the size or position of the disordered region in the sequence. The hydrophobicity profile of the ordered regions of the disordered proteins differ considerably from that of globular proteins implying the role of disordered parts and the significance of hydrophobic interactions in the folding of proteins. The shape asymmetry of the two classes of disordered proteins is determined by calculating the asphercity and shape parameters, derived from the cartesian components of radius of gyration tensor. Disordered proteins of group a are more spherical as compared to the natively unstructured proteins (group b), which are more prolate. Both groups of proteins exhibit similar types of side-chain backbone contacts, as that of the globular proteins. While disordered proteins contains few hydrophobic residues natively unstructured proteins are characterized by a residues of low mean hydrophobicity and high mean net charge.

SUR1 L0 and Kir6.2 M0 can Partner in KATP Gating: Evidences from Diabetogenic Mutations

Neonatal diabetes (ND) mutations in different domains of either KATP subunit overactivate KATP via different mechanisms. Mutations in the ABC core of SUR1 augment its Mg-nucleotide-dependent stimulation of Kir6.2. Mutations at the putative ATP site of Kir6.2 supposedly compromise the inhibitory nucleotide binding. Mutations at the putative gate reportedly stabilize its open state. Many ND mutations map to the putative L0 helix preceding the SUR1 core and to the M0 (‘slide’) helix of Kir6.2. We discovered that L0 controls spontaneous bursting and introduced a model in which L0 and M0 partner in KATP gating (Babenko & Bryan 2003). The model predicted that ND mutations in either amphipathic helix at the membrane-cytosol interface hyperactivate KATP by altering its intrinsic gating kinetics. Here we tested the idea. We compared the effects of severe ND mutations in the middle of either interface helix on spontaneous single-channel kinetics, ATP-inhibition, and Mg-nucleotide stimulation of KATP. We found that each of these mutations decreases the rate of burst termination while increasing the rate of burst initiation, thereby reducing the availability of the closed state with the lowest Kd for inhibitory ligands. This mechanism attenuates ATP inhibition but not Mg-nucleotide stimulation, thereby hyperactivating KATP in vivo, and uncouples sulfonylurea binding from Kir6.2 closure. Thus, in support of our model, perturbing either interface helix disrupts SUR1/Kir6.2 inhibitory coupling. Affinity photolabeling with I-azidoglibenclamide indicated that none of the ND mutations altered the proximity of L0 to M0. But each of these mutations changed the helical hydrophobic moment, implying that rotation of either helix along its axis disrupts L0/M0 interactions. We propose that the two interface helices have been optimized to interact and slide together to stabilize the long-lived closed state of SUR1/Kir6.2 complex.