Helix Macrodipole Research Articles

Effects of Helix Macrodipole and Local Interactions on Catalysis of Acyl Transfer by α-Helical Peptides

The impact of the macrodipole of peptide helices on catalysis was examined using density functional theory calculations. Transition state structures for histidine-containing polyalanine nucleophilic catalysts adding to carbonyls were computed, and the impact of both global and local noncovalent interactions was assessed. Although the peptide macrodipole appears to influence energy barriers, local interactions dominate.

Multiscale Modeling of a Conditionally Disordered pH-Sensing Chaperone

The pH-sensing chaperone HdeA promotes the survival of enteropathogenic bacteria during transit through the harshly acidic environment of the mammalian stomach. At low pH, HdeA transitions from an inactive, folded, dimer to chaperone-active, disordered, monomers to protect against the acid-induced aggregation of periplasmic proteins. Toward achieving a detailed mechanistic understanding of the pH response of HdeA, we develop a multiscale modeling approach to capture its pH-dependent thermodynamics. Our approach combines pKa (logarithmic acid dissociation constant) calculations from all-atom constant pH molecular dynamics simulations with coarse-grained modeling and yields new, atomic-level, insights into HdeA chaperone function that can be directly tested by experiment. “pH triggers” that significantly destabilize the dimer are each located near the N-terminus of a helix, suggesting that their neutralization at low pH destabilizes the helix macrodipole as a mechanism of monomer disordering. Moreover, we observe a non-monotonic change in the pH-dependent stability of HdeA, with maximal stability of the dimer near pH5. This affect is attributed to the protonation Glu37, which exhibits an anomalously high pKa value and is located within the hydrophobic dimer interface. Finally, the pH-dependent binding pathway of HdeA comprises a partially unfolded, dimeric intermediate that becomes increasingly stable relative to the native dimer at lower pH values and displays key structural features for chaperone–substrate interaction. We anticipate that the insights from our model will help inform ongoing NMR and biochemical investigations.

Peroxiredoxin-1 from the Human Hookworm Ancylostoma ceylanicum Forms a Stable Oxidized Decamer and Is Covalently Inhibited by Conoidin A

Hookworms are parasitic nematodes that have a devastating impact on global health, particularly in developing countries. We report a biochemical and structural analysis of a peroxiredoxin from the hookworm Ancylostoma ceylanicum, AcePrx-1. Peroxiredoxins provide antioxidant protection and act as signaling molecules and chaperones. AcePrx-1 is expressed in adult hookworms and can be inactivated by 2,3-bis(bromomethyl)quinoxaline-1,4-dioxide (conoidin A). Conoidin A inactivates AcePrx-1 by alkylating or crosslinking the catalytic cysteines,while maintaining the enzyme in the "locally unfolded" conformation. Irreversible oxidation of the resolving cysteine may contribute additional inhibitory activity. A crystal structure of oxidized AcePrx-1 reveals a disulfide-linked decamer. A helix macrodipole near the active site increases the reactivity of the catalytic cysteines to conoidin A. This work demonstrates the promise of conoidin compounds as probes to evaluate peroxiredoxins as drug targets in human parasites.

Understanding the pKaof Redox Cysteines: The Key Role of Hydrogen Bonding

Many cellular functions involve cysteine chemistry via thiol-disulfide exchange pathways. The nucleophilic cysteines of the enzymes involved are activated as thiolate. A thiolate is much more reactive than a neutral thiol. Therefore, determining and understanding the pK(a)s of functional cysteines are important aspects of biochemistry and molecular biology with direct implications for redox signaling. Here, we describe the experimental and theoretical methods to determine cysteine pK(a) values, and we examine the factors that control these pK(a)s. Drawing largely on experience gained with the thioredoxin superfamily, we examine the roles of solvation, charge-charge, helix macrodipole, and hydrogen bonding interactions as pK(a)-modulating factors. The contributions of these factors in influencing cysteine pK(a)s and the associated chemistry, including the relevance for the reaction kinetics and thermodynamics, are discussed. This analysis highlights the critical role of direct hydrogen bonding to the cysteine sulfur as a key factor modulating the equilibrium between thiol S-H and thiolate S(-). This role is easily understood intuitively and provides a framework for biochemical functional insights.

Playing with Peptides: How to Build a Supramolecular Peptide Nanostructure by Exploiting Helix···Helix Macrodipole Interactions

A novel method to build bicomponent peptide self-assembled monolayers (SAMs) has been developed, by exploiting helix···helix macrodipole interactions. In this work, a peptide-based self-assembled monolayer composed of two helical peptides was immobilized on a gold surface. Specifically, a pyrene-containing octapeptide, devoid of any sulfur atom (A8Pyr), and a hexapeptide, functionalized at the N-terminus with (S,R) lipoic acid, for binding to gold substrates (SSA4WA) via a Au-S linkage, have been employed. Both peptides investigated attain a helical structure, because they are almost exclusively formed by strongly folding inducer C(α)-tetrasubstituted α-amino acids. We demonstrate that the two peptides generate a stable supramolecular nanostructure (a densely packed bicomponent peptide monolayer), where A8Pyr is incorporated into the SSA4WA palisade by exploiting helix···helix macrodipole interactions. The presence of both peptides on the gold surface was investigated by spectroscopic and electrochemical techniques, while the morphology of the monolayer was analyzed by ultra high-vacuum scanning tunnelling microscopy. The composition of the bicomponent SAM on the surface was studied by a combination of electrochemical and spectroscopic techniques. In particular, the amount of Au-S linkages from the sulfur-containing peptides was quantified from reductive desorption of the peptide-based SAM, while the amount of A8Pyr was estimated by fluorescence spectroscopy. The antiparallel orientation of the A8Pyr and SSA4WA peptide chains minimizes the interaction energy between the helix dipoles, suggesting that this kind of electrostatic phenomenon is the driving force that stabilizes the bicomponent SAM.

N-Protonated Isomers as Gateways to Peptide Ion Fragmentation

According to the popular "mobile proton model" for peptide ion fragmentation in tandem mass spectrometry, peptide bond cleavage is typically preceded by intramolecular proton transfer from basic sites to an amide nitrogen in the backbone. If the intrinsic barrier to dissociation is the same for all backbone sites, the fragmentation propensity at each amide bond should reflect the stability of the corresponding N-protonated isomer. This hypothesis was tested by using ab initio and force-field computations on several polyalanines and Leu-enkephalin. The results agree acceptably with experimental reports, supporting the hypothesis. It was found that backbone N-protonation is most favorable near the C-terminus. The preference for C-terminal N-protonation, which is stronger for longer polyalanines, may be understood in terms of the well known "helix macrodipole" in the corresponding helical conformations. The opposite stability trend is found for peptides constrained to be linear, which is initially surprising but turns out to be consistent with the reversed direction of the macrodipole in the linear conformation.

Double-brush Langmuir–Blodgett monolayers of α-helical diblock copolypeptides

The synthesis of amphiphilic diblock copolypeptides consisting of poly(α-l-glutamic acid) (PLGA) and poly(γ-methyl-l-glutamate-ran-γ-stearyl-l-glutamate) with 30mol % of stearyl substituents (PMLGSLG) and their monolayer behavior at the air–water interface have been studied. PLGA-b-PMLGSLG was synthesized via a diblock copolymer precursor consisting of poly(γ-tert-butyl-l-glutamate) (PtBuLG) and PMLGSLG blocks, with the tert-butyl group as a mild acid-labile protecting group for the carboxylic acid. The polymerization conditions were found to influence the α-helix to β-sheet content ratio and can be tuned to significantly enhance the diblock copolypeptide helicity. Purely α-helical PtBuLG-b-PMLGSLG diblock copolymers were successfully prepared. After removal of the tert-butyl group, the study of the PLGA-b-PMLGSLG amphiphilic diblock copolymers in Langmuir monolayers and Langmuir–Blodgett films demonstrated the formation of a stable α-helical double-brush structure, with the helices tilted away from the substrate surface. These double-brush monolayers combine the unique properties arising from the unidirectionally aligned helix macrodipole and the liquid-like features of the side chain mantle of the PMLGSLG block. Such systems are promising for thin film applications requiring incorporation and orientation of bio- and optical molecules.

Secbase: Database Module To Retrieve Secondary Structure Elements with Ligand Binding Motifs

Secbase is presented as a novel extension module of Relibase. It integrates the information about secondary structure elements into the retrieval facilities of Relibase. The data are accessible via the extended Relibase user interface, and integrated retrieval queries can be addressed using an extended version of Reliscript. The primary information about alpha-helices and beta-sheets is used as provided by the PDB. Furthermore, a uniform classification of all turn families, based on recent clustering methods, and a new helix assignment that is based on this turn classification has been included. Algorithms to analyze the geometric features of helices and beta-strands were also implemented. To demonstrate the performance of the Secbase implementation, some application examples are given. They provide new insights into the involvement of secondary structure elements in ligand binding. A survey of water molecules detected next to the N-terminus of helices is analyzed to show their involvement in ligand binding. Additionally, the parallel oriented NH groups at the alpha-helix N-termini provide special binding motifs to bind particular ligand functional groups with two adjacent oxygen atoms, e.g., as found in negatively charged carboxylate or phosphate groups, respectively. The present study also shows that the specific structure of the first turn of alpha-helices provides a suitable explanation for stabilizing charged structures. The magnitude of the overall helix macrodipole seems to have no or only a minor influence on binding. Furthermore, an overview of the involvement of secondary structure elements with the recognition of some important endogenous ligands such as cofactors shows some distinct preference for particular binding motifs and amino acids.

Gas-Phase Acidities of Cysteine-Polyalanine Peptides I: A3,4CSH and HSCA3,4

The gas-phase acidities of four cysteine-polyalanine peptides, A(3,4)CSH and HSCA(3,4), were determined using the extended Cooks kinetic method with full entropy analysis. A triple-quadrupole mass spectrometer with an electrospray interface was employed for the experimental study. The ion activation was achieved via collision-induced dissociation (CID) experiments. The deprotonation enthalpies (Delta(acid)H) of the peptides were determined to be 332.2 +/- 2.0 kcal/mol (A(3)CSH), 325.9 +/- 2.0 kcal/mol (A(4)CSH), 319.3 +/- 3.0 kcal/mol (HSCA(3)), and 319.2 +/- 4.0 kcal/mol (HSCA(4)). The deprotonation entropies (Delta(acid)S) of the peptides were estimated based on the entropy term (Delta(DeltaS)) and the deprotonation entropies of the reference acids. By using the deprotonation enthalpies and entropies, the gas-phase acidities (Delta(acid)G) of the peptides were derived: 325.0 +/- 2.0 kcal/mol (A(3)CSH), 320.2 +/- 2.0 kcal/mol (A(4)CSH), 316.3 +/- 3.0 kcal/mol (HSCA(3)), and 315.4 +/- 4.0 kcal/mol (HSCA(4)). Conformations and energetic information of the peptides were calculated through simulated annealing (Tripos), geometry optimization (AM1), and single-point energy calculations (B3LYP/6-31+G(d)), respectively. The calculated theoretical deprotonation enthalpies (Delta(acid)H) of 334.2 kcal/mol (A(3)CSH), 327.7 kcal/mol (A(4)CSH), 320.6 kcal/mol (HSCA(3)), and 318.6 kcal/mol (HSCA(4)) are in good agreement with the experimentally determined values. Both the experimental and computational studies suggest that the two N-terminal cysteine peptides, HSCA(3,4), are significantly more acidic than the corresponding C-terminal ones, A(3,4)CSH. The high acidities of the former are likely due to the helical conformational effects for which the thiolate anion may be strongly stabilized by the interaction with the helix macrodipole.

Thermodynamics of Membrane Partitioning and Self-association of Transmembrane Helices: Impact of Lipid Composition

Membrane partitioning and self-association of transmembrane helices (TMHs) have been extensively investigated using model TMHs to elucidate the driving forces of membrane protein folding. We designed the inert hydrophobic model TMH (AALALAA)3 to obtain thermodynamic parameters related to the steps. The formation of the antiparallel dimer was detected by fluorescence resonance energy transfer between fluorescent labeled peptides [1]. Stronger dimerization was observed in thicker membranes and at lower temperatures (ΔG = −9 – −26 kJ mol−1), driven by large negative ΔH values (−18 – −80 kJ mol−1). The enthalpy changes for helix-helix interaction can be well explained by electrostatic interaction between helix macrodipoles in different dielectric environments. The incorporation of cholesterol and PE also facilitated the dimerization by large negative ΔH values. The partitioning process was also investigated based on the transfer of the helix between vesicles [2]. Under hydrophobic mismatch conditions up to ∼7 Å, the helix partitioning became unfavorable up to +7 kJ mol−1, hampered by an increase in entropic (up to +20 kJ mol−1) and enthalpic (up to +66 kJ mol−1) terms in thinner and thicker membranes, respectively. The obtained thermodynamic parameters were reasonably explained assuming that hydrophobic mismatch induces aqueous exposure or membrane burial of the helix termini, respectively. I also present a design of a water-soluble TMH for the direct measurement of the partitioning process. Furthermore, I will introduce a novel method for quick fluorescent labeling of membrane proteins to detect TMH interactions in living cell membranes [3]. [1] Y. Yano and K. Matsuzaki, Biochemistry 45, 3370 (2006). [2] Y. Yano et al., Biochemistry 45, 3379 (2006). [3] Y. Yano et al., ACS Chem. Biol. 6, 341 (2008).

X-ray Structures of the Three Lactococcus lactis Dihydroxyacetone Kinase Subunits and of a Transient Intersubunit Complex

Bacterial dihydroxyacetone (Dha) kinases do not exchange the ADP for ATP but utilize a subunit of the phosphoenolpyruvate carbohydrate phosphotransferase system for in situ rephosphorylation of a permanently bound ADP-cofactor. Here we report the 2.1-angstroms crystal structure of the transient complex between the phosphotransferase subunit DhaM of the phosphotransferase system and the nucleotide binding subunit DhaL of the Dha kinase of Lactococcus lactis, the 1.1-angstroms structure of the free DhaM dimer, and the 2.5-angstroms structure of the Dha-binding DhaK subunit. Conserved salt bridges and an edge-to-plane stacking contact between two tyrosines serve to orient DhaL relative to the DhaM dimer. The distance between the imidazole Nepsilon2 of the DhaM His-10 and the beta-phosphate oxygen of ADP, between which the gamma-phosphate is transferred, is 4.9 angstroms. An invariant arginine, which is essential for activity, is appropriately positioned to stabilize the gamma-phosphate in the transition state. The (betaalpha)4alpha fold of DhaM occurs a second time as a subfold in the DhaK subunit. By docking DhaL-ADP to this subfold, the nucleotide bound to DhaL and the C1-hydroxyl of Dha bound to DhaK are positioned for in-line transfer of phosphate.

The barrier for proton transport in aquaporins as a challenge for electrostatic models: The role of protein relaxation in mutational calculations

The origin of the barrier for proton transport through the aquaporin channel is a problem of general interest. It is becoming increasingly clear that this barrier is not attributable to the orientation of the water molecules across the channel but rather to the electrostatic penalty for moving the proton charge to the center of the channel. However, the reason for the high electrostatic barrier is still rather controversial. It has been argued by some workers that the barrier is due to the so-called NPA motif and/or to the helix macrodipole or to other specific elements. However, our works indicated that the main reason for the high barrier is the loss of the generalized solvation upon moving the proton charge from the bulk to the center of the channel and that this does not reflect a specific repulsive electrostatic interaction but the absence of sufficient electrostatic stabilization. At this stage it seems that the elucidation and clarification of the origin of the electrostatic barrier can serve as an instructive test case for electrostatic models. Thus, we reexamine the free-energy surface for proton transport in aquaporins using the microscopic free-energy perturbation/umbrella sampling (FEP/US) and the empirical valence bond/umbrella sampling (EVB/US) methods as well as the semimacroscopic protein dipole Langevin dipole model in its linear response approximation version (the PDLD/S-LRA). These extensive studies help to clarify the nature of the barrier and to establish the "reduced solvation effect" as the primary source of this barrier. That is, it is found that the barrier is associated with the loss of the generalized solvation energy (which includes of course all electrostatic effects) upon moving the proton charge from the bulk solvent to the center of the channel. It is also demonstrated that the residues in the NPA region and the helix dipole cannot be considered as the main reasons for the electrostatic barrier. Furthermore, our microscopic and semimacroscopic studies clarify the problems with incomplete alternative calculations, illustrating that the effects of various electrostatic elements are drastically overestimated by macroscopic calculations that use a low dielectric constant and do not consider the protein reorganization. Similarly, it is pointed out that microscopic potential of mean force calculations that do not evaluate the electrostatic barrier relative to the bulk water cannot be used to establish the origin of the electrostatic barrier. The relationship between the present study and calculations of pK(a)s in protein interiors is clarified, pointing out that approaches that are applied to study the aquaporin barrier should be validated by pK(a)s calculations. Such calculations also help to clarify the crucial role of solvation energies in establishing the barrier in aquaporins.

Measurement of Thermodynamic Parameters for Hydrophobic Mismatch 1: Self-Association of a Transmembrane Helix†

Membrane partitioning and self-association of transmembrane helices are crucial thermodynamic steps for membrane protein folding, although experimental difficulties have hampered quantitative estimations of related thermodynamic parameters, especially in lipid bilayer environments. This article reports for the first time, the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the formation of the antiparallel dimer of the inert hydrophobic model transmembrane helix X-(AALALAA)(3)-Y (X = 7-nitro-2-1, 3-benzoxadiazol-4-yl (NBD) and Y = NH(2) (I) or X = Ac and Y = NHCH(2)CH(2)-S-N-[4-[[4-(dimethylamino)phenyl]azo]phenyl]maleimide (DABMI) (II)) in dimonounsaturated phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 5-55 degrees C, as evaluated by fluorescence resonance energy transfer from I to II. Stronger dimerization was observed in thicker membranes and at lower temperatures (DeltaG = -9 to -26 kJ mol(-)(1)), driven by large negative DeltaH values (-18 to -80 kJ mol(-)(1)). Fourier transform infrared-polarized spectroscopy revealed that the peptide formed a stable transmembrane helix with an orientation angle of approximately 15 degrees in all bilayers without significant effects on lipid structures, suggesting that the depth to which the helix termini penetrate changes depending on the degree of hydrophobic mismatch. The enthalpy changes for helix-helix interactions can be well explained by the electrostatic interactions between helix macrodipoles in different dielectric environments. The new concept of dipole-dipole interaction as a basic driving force of helix dimerization will become a basis for understanding the structural and functional modifications in response to hydrophobic mismatch.

Measurement of Thermodynamic Parameters for Hydrophobic Mismatch 2: Intermembrane Transfer of a Transmembrane Helix

Hydrophobic matching between proteins and lipids is essential for the thermodynamic stability of integral membrane proteins. However, there is no direct thermodynamic information available about the intermembrane transfer of proteins between membranes with different hydrophobic thicknesses, which is crucial for understanding hydrophobic mismatch. This article reports the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the intermembrane transfer of the inert hydrophobic model transmembrane helix NBD-(AALALAA)(3)-NH(2) (NBD: 7-nitro-2-1,3-benzoxadiazol-4-yl), which is exchangeable between vesicles, from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to dimonounsaturated-phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 37-58 degrees C. The transfer free energies were calculated from equilibrium values of the extent of helix transfer from donor to acceptor lipid vesicles, as monitored by a decrease in fluorescence resonance energy transfer from the NBD group to a lipid-labeled Rhodamine in the donor upon transfer to the quencher-free acceptor. Under hydrophobic mismatch conditions up to approximately 7 A, the helix partitioning became unfavorable up to +7 kJ mol(-)(1), hampered by an increase in entropic (up to +20 kJ mol(-)(1)) and enthalpic (up to +66 kJ mol(-)(1)) terms in thinner and thicker membranes, respectively. Together with the results that H/D exchange at the membrane interface was accelerated in thinner membranes the obtained thermodynamic parameters were reasonably explained assuming that hydrophobic mismatch induces aqueous exposure or membrane burial of the helix termini, resulting in excess energies originating from the hydration of terminal hydrophobic residues or the unfavorable Born energy of terminal partial charges of the helix macrodipole.

Hydrogen bonding is the prime determinant of carboxyl pKa values at the N‐termini of α‐helices

Experimentally determined mean pK(a) values of carboxyl residues located at the N-termini of alpha-helices are lower than their overall mean values. Here, we perform three types of analyses to account for this phenomenon. We estimate the magnitude of the helix macrodipole to determine its potential role in lowering carboxyl pK(a) values at the N-termini. No correlation between the magnitude of the macrodipole and the pK(a) values is observed. Using the pK(a) program propKa we compare the molecular surroundings of 18 N-termini carboxyl residues versus 233 protein carboxyl groups from a previously studied database. Although pK(a) lowering interactions at the N-termini are similar in nature to those encountered in other protein regions, pK(a) lowering backbone and side-chain hydrogen bonds appear in greater number at the N-termini. For both Asp and Glu, there are about 0.5 more hydrogen bonds per residue at the N-termini than in other protein regions, which can be used to explain their lower than average pK(a) values. Using a QM-based pK(a) prediction model, we investigate the chemical environment of the two lowest Asp and the two lowest Glu pK(a) values at the N-termini so as to quantify the effect of various pK(a) determinants. We show that local interactions suffice to account for the acidity of carboxyl residues at the N-termini. The effect of the helix dipole on carboxyl pK(a) values, if any, is marginal. Backbone amide hydrogen bonds constitute the single biggest contributor to the lowest carboxyl pK(a) values at the N-termini. Their estimated pK(a) lowering effects range from about 1.0 to 1.9 pK(a) units.

Electrostatics of Ion Stabilization in a ClC Chloride Channel Homologue from Escherichia coli

The structural determinants of electrostatics of ion stabilization within EcClC, a ClC-type chloride channel homologue from Escherichia coli, are studied using a continuum dielectric approximation. Specifically, the ion occupancy is investigated in the wild-type protein and a mutant thereof, and the contribution to the electrostatic binding free energy of local and non-local interactions is characterized at the single-residue level. This analysis shows that, in spite of the desolvation cost and the strong ion–ion repulsion, all previously reported binding sites can be occupied simultaneously. The stabilizing effect of the protein arises from hydrogen bonding as well as from longer-range favorable interactions, such as with the strictly conserved Lys131 side-chain. The latter is involved in the stabilization of the conserved GSGIP motif that delimits two of the binding sites. Interestingly, an additional low-affinity binding site, mediated by a structurally analogous motif including the side-chain of Arg340, can be identified on the extracellular side of the permeation pathway. Finally, it is shown that, in contrast to K-channels, and in analogy to the SBP/PBP sulfate/phosphate-binding proteins, the contribution of helix macrodipoles to chloride binding in EcClC is only marginal.

Anhydrous Polyproline Helices and Globules

Ion mobility/time-of-flight methods and molecular modeling calculations have been used to examine the conformations of a range of polymer lengths and charge states of polyproline peptides, [Pron + zH]z+ (n = 3−56; z = 1−6). Ions formed from 1-propanol solutions {[Pron + H]+ (n = 5−11) and [Pron + 2H]2+ (n = 10−22)} favor extended forms of the classical polyproline I helix. In these conformers, all proline residues are in the cis configuration, and protonation at the N-terminus allows hydrogen bonds to be formed with backbone carbonyl groups of the second and third proline residues in each polymer. Protonation of this all-cis form at the N-terminus also stabilizes the helix macrodipole. Singly charged ions formed from aqueous solutions favor globular and hairpin-like conformers that contain both cis- and trans-proline residues. Higher charge state ions (z = 3−6) formed from aqueous solutions favor relatively extended conformations, although these are not as extended as the polyproline II structural limit. ...

Pi-helix preference in unsolvated peptides.

Ion mobility measurements have been used to examine helix formations in the gas phase for a series of alanine/glycine-based peptides that incorporate a glutamic acid (E) and lysine (K) at various positions along the backbone. Incorporation of an EK pair lowers the percent helix for all positions (presumably because hydrogen bonding between the backbone and the E and K side chains stabilize the nonhelical globular conformations). The largest percent helix is found when the EK pair is in an i,i+5 arrangement, which suggests that the preferred helical conformation for these peptides is a pi-helix. This conclusion is supported by comparison of cross sections deduced from the ion-mobility measurements to average cross sections calculated for conformations obtained from molecular dynamics simulations. The glutamic acid and lysine may form an ion pair that is stabilized by interactions with the helix macro-dipole.

Importance of alpha-helix N-capping motif in stabilization of betabetaalpha fold.

FSD-1 (full sequence design 1) is a protein folded in a betabetaalpha motif, designed on the basis of the second zinc finger domain of Zif268 by a substitution of its metal coordination site with a hydrophobic core. In this work, we analyzed the possibility of introducing the DNA recognition motif of the template zinc finger (S(13)RSDH(17)) into FSD-1 sequence in order to obtain a small DNA-binding module devoid of cross-link(s) or metal cofactors. The hybrid protein was unfolded, as judged by CD and NMR criteria. To reveal the role of each of the five amino acids, which form the N-capping motif of the alpha-helix, we analyzed conformational and stability properties of eight FSD-1 mutants. We used a shielded methyl group of Leu 18 and a CD signal at 215 nm as a convenient measure of the folded state. Glu 17-->His substitution at the N(3) in S(13)NEKE(17) variant decreased the folded structure content from 90% to 25% (equivalent to 1.8 kcal * mole(-1) destabilization) by disruption of N-capping interactions, and had the most significant effect among single mutants studied here. The N(cap) Asn 14 substitution with Arg considerably decreased stability, reducing structure content from 90% to 40% (1.4 kcal * mole(-1) destabilization) by disruption of a helix-capping hydrogen bond and destabilization of a helix macrodipole. The N(1) Glu 15-->Ser mutation also produced a considerable effect (1.0 kcal * mole(-1) destabilization), again emphasizing the significance of electrostatic interactions in alpha-helix stabilization.

Effect of the helix macrodipole on surface-initiated N-carboxyanhydride polymerization on gold.

The right moment for polymerization: Preformed helical rods on gold surfaces are shown to control N-carboxyanhydride polymerization through the helix-macrodipole interaction (see picture; polymer length depends on the direction of the dipole), and have allowed the successful preparation of densely, perpendicularly bound helical brushes on gold surfaces.