The role of local tight packing of hydrophobic groups in β-structure

An analysis of the tendency of hydrophobic groups to tight packing on the surface of β-sheets based on well-known parameters of β-sheets and hydrophobic groups was conducted. This analysis shows the existence of very limited numbers and clearly outlined architecture families of regular parts for the majority of β-structure-containing domains. Each family of architecture strongly depends on the number of β-strands in the pure β-domains and on the existence and number of additional α-helixes and on the mutual arrangements β-strands and α-helixes along the chain in mixed α/β-domains. This paper demonstrates that the tendency of hydrophobic groups to the local tight packing on the surface of β-sheets is probably the main reason for the twist of β-sheets. © 1993 Wiley-Liss, Inc.     

Kinetics of coil to α-helix to β-structure transitions of poly(L-ornithine) in low concentrations of sodium dodecyl sulfate

Conformational changes of poly(L-ornithine) [(Orn)_n] were studied in a sodium dodecyl sulfate (NaDodSO₄) solution by CD. (Orn)_n adopted an unstable and a stable helical structure below and above the NaDodSO₄ concentration range where β-structure was favored, respectively. CD stopped-flow was used to monitor the transitions from coil to the unstable helix, from the helix to β-structure, and from coil to β-structure. Only the rate of the helix to β-structure transition was accelerated by an increase in NaDodSO₄ concentration, whereas the rates of the others were independent of NaDodSO₄ concentration. The fractions of coil, α-helix, and β-structure in each conformation of (Orn)_n caused by NaDodSO₄ were computed by simulating a mixed spectrum of typical CD spectra for these structures to the experimentally obtained spectrum. The contents of the unstable and stable helical structures were less than 50 and 73%, respectively.     

Synthetic studies toward labionin,a new α,α‐disubstituted amino acid from type III lantibiotic labyrinthopeptin A2

The labyrinthopeptins are a new class of lantibiotics containing two identical quaternary α,α‐disubstituted amino acids, named labionin (Lab). The synthetic formation of this unique structural feature represents the key step in the total synthesis of these polycyclic peptides. In this report we describe the synthesis of an orthogonally protected α,α‐disubstituted amino acid building block serving as labionin precursor for the future assembly of labyrinthopeptin A2 and of other labyrinthopeptin derivatives. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A dihedral angle-vicinal proton coupling constant correlation for the α–β bond of amino acid residues

A coupling constant-dihedral angle correlation for the H? Cα? Cβ? H system of amino acid residues in peptides has been derived from a set of model compounds covering the full range of dihedral angles. The expression obtained, J = 11.0 cos₂ θ ?1.4 cos θ + 1.6 sin₂θ, is close to those already used in pmr studies of peptide conformation, and provides a firmer foundation for them. A factor limiting the precision of this and other “Karplus relations” is illustrated.     

Assembly,trafficking and function of α1β2γ2 GABAA receptors are regulated by N‐terminal regions,in a subunit‐specific manner

GABA_A receptors are pentameric ligand‐gated ion channels that mediate inhibitory fast synaptic transmission in the central nervous system. Consistent with recent pentameric ligand‐gated ion channels structures, sequence analysis predicts an α‐helix near the N‐terminus of each GABA_A receptor subunit. Preceding each α‐helix are 8–36 additional residues, which we term the N‐terminal extension. In homomeric GABA_C receptors and nicotinic acetylcholine receptors, the N‐terminal α‐helix is functionally essential. Here, we determined the role of the N‐terminal extension and putative α‐helix in heteromeric α1β2γ2 GABA_A receptors. This role was most prominent in the α1 subunit, with deletion of the N‐terminal extension or further deletion of the putative α‐helix both dramatically reduced the number of functional receptors at the cell surface. Conversely, deletion of the β2 or γ2 N‐terminal extension had little effect on the number of functional cell surface receptors. Additional deletion of the putative α‐helix in the β2 or γ2 subunits did, however, decrease both functional cell surface receptors and incorporation of the γ2 subunit into mature receptors. In the β2 subunit only, α‐helix deletions affected GABA sensitivity and desensitization. Our findings demonstrate that N‐terminal extensions and α‐helices make key subunit‐specific contributions to assembly, consistent with both regions being involved in inter‐subunit interactions.

     

Conformations of peptides containing a chiral cyclic α, α‐disubstituted α‐amino acid within the sequence of Aib residues

A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac₅c^dOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and ¹H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac₅c^dOM]‐(Aib)₄‐OEt ( 1) and Cbz‐(Aib)₄‐[(S,S)‐Ac₅c^dOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 3₁₀‐helical structure was present in 1 and that a right‐handed (P) 3₁₀‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Vibrational spectroscopy of L-valyl-glycyl-glycine,a parallel-chain β-structure

Bands in the ir and Raman spectra of L -valyl-glycyl-glycine (VGG) and VGG-ND have been assigned on the basis of a normal mode analysis of the known parallel-chain β-structure of this tripeptide. Amide I, II, III, and V mode shifts are obtained by the interactions of dipole derivatives in symmetry coordinates, referred to as dipole derivative coupling. These derivatives, obtained from ab initio studies, are also used to calculate ir intensities of amide I, II, and V modes. The agreement between predicted and observed frequencies and intensities is very good, providing confidence in the application of our force fields to the calculation of the vibrational modes of the general parallel-chain β-sheet structure (following paper).     

Identification of a chemotactic epitope in human transforming growth factor-β1 spanning amino acid residues 368–374

TGF-b?1 plays a critical role in inflammatory and repair processes due in part to its ability to provide a potent chemotactic stimulus for inflammatory cells such as neutrophils and monocytes and for fibroblasts which initiate the fibrogenic response. In the present study, we have used synthetic oligopeptides representing the amino acid sequence of the 12.1 kDa monomer of human TGF-b?1 in an effort to identify a chemotactic epitope on the molecule. A seven residue peptide containing residues 368-374, Val Tyr Tyr Val Gly Arg Lys, was demonstrated to be capable of inducing chemotactic migration of human peripheral blood neutrophils, monocytes, monocyte leukemia cell line THP-1, and infant foreskin fibroblasts. Furthermore, larger peptides from the carboxy-terminal portion of TGF-b?1 that contained residues 368–374 also induced migration of these cell types. None of the peptides representing the complete amino acid of TGF-b?1 monomer were able to compete with [¹²⁵I]hrTGF-b?1 for binding to TGF-b? cell surface receptors or fibroblasts or THP-1 cells. Implications of these observations are discussed. © 1995 Wiley-Liss, Inc.     

Low-sulfur proteins from α-keratins. Interrelationships between their amino acid compositions, α-helix contents,and the supercontraction of the parent keratin

S-Carboxymethyl (SCM) kerateine preparations from a range of keratins were fractionated by acid precipitation into low-sulfur (SCMKA) and high-sulfur (SCMKB) fractions. Amiono acid analyses and optical rotatory dispersion measurements on the SCMKA fractions from different keratins indicated that the proportion of α-helical material (30–64%) increased with increasing leucine and glutamic acid contents and decreased with increasing SCM-cysteine and proline contents. It is shown that these variations in α-helix contents are due mainly to differences in the conformations of the principal components of the SCMKA fractions, although there is also a smaller contribution arising from contamination with varying amounts of non helical components. The thiol + disulfide contents for the parent keratins were measured and compared with the SCM -cysteine contents of the solubilized fractions. In general the SCM-cysteine contents of the SCMKA fractions increased with increasing thiol + disulfide content of the parent keratin, but these values were not directly proportional. The extent of total supercontraction in 8M LiBr solution was measured for the parent keratins and shown to bear no significant relationship to their disulfide contents. From the extents of first-stage and total supercontractions the disulfide contents of the contractile structures in the fibers were calculated according to a model described previously and compared with the analytical values for the SCM -cysteine contents of the SCMKA and SCMKB fraction. Data for both fractions were consistent with the model in that they were in each case related to the calculated values by a constant factor. However, because of simplifying assumptions in the model, it was not possible to identity positively one or other of these protein fractions with the contractile elements in the fiber.     

Selective inhibition of 11β-hydroxysteroid dehydrogenase 1 by 18α-glycyrrhetinic acid but not 18β-glycyrrhetinic acid

Elevated cortisol concentrations have been associated with metabolic diseases such as diabetes type 2 and obesity. 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1, catalyzing the conversion of inactive 11-ketoglucocorticoids into their active 11β-hydroxy forms, plays an important role in the regulation of cortisol levels within specific tissues. The selective inhibition of 11β-HSD1 is currently considered as promising therapeutic strategy for the treatment of metabolic diseases. In recent years, natural compound-derived drug design has gained considerable interest. 18β-glycyrrhetinic acid (GA), a metabolite of the natural product glycyrrhizin, is not selective and inhibits both 11β-HSD1 and 11β-HSD2. Here, we compare the biological activity of 18β-GA and its diastereomer 18α-GA against the two enzymes in lysates of transfected HEK-293 cells and show that 18α-GA selectively inhibits 11β-HSD1 but not 11β-HSD2. This is in contrast to 18β-GA, which preferentially inhibits 11β-HSD2. Using a pharmacophore model based on the crystal structure of the GA-derivative carbenoxolone in complex with human 11β-HSD1, we provide an explanation for the differences in the activities of 18α-GA and 18β-GA. This model will be used to design novel selective derivatives of GA.     

Rap1 controls activation of the αMβ2 integrin in a talin‐dependent manner

The small GTPase Rap1 and the cytoskeletal protein talin regulate binding of C3bi‐opsonised red blood cells (RBC) to integrin α_Mβ₂ in phagocytic cells, although the mechanism has not been investigated. Using COS‐7 cells transfected with α_Mβ₂, we show that Rap1 acts on the β₂ and not the α_M chain, and that residues 732–761 of the β₂ subunit are essential for Rap1‐induced RBC binding. Activation of α_Mβ₂ by Rap1 was dependent on W747 and F754 in the β₂ tails, which are required for talin head binding, suggesting a link between Rap1 and talin in this process. Using talin1 knock‐out cells or siRNA‐mediated talin1 knockdown in the THP‐1 monocytic cell line, we show that Rap1 acts upstream of talin but surprisingly, RIAM knockdown had little effect on integrin‐mediated RBC binding or cell spreading. Interestingly, Rap1 and talin influence each other's localisation at phagocytic cups, and co‐immunoprecipitation experiments suggest that they interact together. These results show that Rap1‐mediated activation of α_Mβ₂ in macrophages shares both common and distinct features from Rap1 activation of α_IIbβ₃ expressed in CHO cells. J. Cell. Biochem. 111: 999–1009, 2010. © 2010 Wiley‐Liss, Inc.     

Lymphocyte transendothelial migration toward smooth muscle cells in interleukin-1 β-stimulated co-cultures is related to fibronectin interactions with α4β1 and α5β1 integrins

We previously reported infiltration of immune-inflammatory cells in coronary arteries from cardiac allografts, associated with increased endothelial and smooth muscle cell fibronectin synthesis regulated by interleukin (IL)-1b?. We now investigate, using a porcine endothelial-smooth muscle cell co-culture system, whether IL-1b?-stimulated fibronectin production is functionally important in lymphocyte transendothelial migration. Lymphocytes were harvested from porcine peripheral blood and, in the unactivated state or following activation with phorbol myristic acetate (PMA) and IL-2, were characterized by fluorescence-activated cell sorter (FACS) analysis and added to a confluent endothelial monolayer on the upper chamber of a transwell system. Endothelial cells, as well as smooth muscle cells (in the bottom of the chamber), were stimulated with IL-1b?. Then transendothelial lymphocyte migration was determined in the presence of CS1 and RGD (fibronectin) peptides, blocking α₄b?₁ and α₅b?₁ integrin receptors on lymphocyte surfaces, respectively. A 55-70% inhibition of lymphocyte migration was observed when compared to control peptides. The combination of CS1 and RGD peptides did not significantly enhance the inhibitory effect of either peptide alone. A similar decrease in lymphocyte transendothelial migration toward smooth muscle cells was documented using a monoclonal antibody to cellular fibronectin. Furthermore, using smooth muscle cell conditioned medium; we reproduced the enhanced transendothelial lymphocyte migration as well as the inhibition with blocking peptides or fibronectin antibodies. Our data suggest that cytokine-mediated fibronectin synthesis in vascular cells recruits inflammatory cells through interactions of specific peptides with cell surface α₄b?₁ α₅b?₁ integrins. © 1995 Wiley-Liss, Inc.     

Relative stability of the α-helix of deuterated poly(γ-benzyl-L-glutamate)

The coil-to-helix transition temperatures of hydrogen bearing and deuterated poly(γ-benzyl-L -glutamate) in 1,3-dichlorotetrafluoroacetone/H₂O and/D₂O mixtures, respectively, have been determined. Together with previously obtained data for the conformational transition of this polypeptide in normal and deuterated dichloroacetic acid, these results have been used in an analysis of the effect of deuterium substitution on the intrinsic stability of the α-helical form of poly(γ-benzyl-L -glutamate). The findings, consistent for both solvent systems, showed that the deuterated polypeptide is some 5% more stable than the normal protonated poly(γ-benzyl-L -glutamate), while the polypeptide-active solvent interaction enthalpy is also slightly increased by deuterium substitution in the respective molecules. A consideration of available data for poly(β-benzyl-L -aspartate) reveals an anomaly with respect to the present analysis.     

Stabilizing effects of 2-methylalanine residues on β-turns and α-helices

¹³C-, ¹H-nmr, CD, and x-ray crystallography revealed β-turns of type III for Boc-Gly-L-Ala-Aib-OMe, Boc-L-Ala-Aib-L-Ala-OMe; the 3₁₀-helix for Boc-Aib-L-Ala-Aib-L-Ala-Aib-OMe; and antiparallel arranged α-helices for Boc-L-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib-Ala-OMe. An N-terminal rigid α-helical segment is found in the polypeptide antibiotics alamethicin, suzukacillin, and trichotoxin. The α-helix dipole is essential for their voltage-dependent pore formation in lipid bilayer membranes, which is explained by a flip-flop gating mechanism based on dipole–dipole interactions of parallel and antiparallel arranged α-helices within oligomeric structures.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

Vibrational approach to the dynamics of an α-helix

The dynamics of a finite α-helix have been studied in the harmonic approximation by a vibrational analysis of the atomic motions about their equilibrium positions. The system were represented by an empirical potential energy function, and all degrees of freedom (bond lengths, bond angles, and torsional angles) were allowed to vary. The complete results were compared with a more restrictive model in which the peptide dihedral angle was kept rigid; also, a model potential excluding hydrogen bonds was examined. Thermal fluctuations in the backbone dihedral angles ? and ψ are 12° to 15°. The fluctuations of adjacent dihedral angles are highly correlated, and the correlation pattern is affected by the flexibility of the peptide dihedral angle. Time-dependent autocorrelations in the motion of ? and ψ appear to decay due to dephasing in less than 1 psec, while the motions of the carbonyl oxygen and amide hydrogens out of the peptide plane are more harmonic. Length fluctuations have been evaluated and exhibit a strong end effect; the calculated elastic modulus is in agreement with other values. Rigid and adiabatic total energy surfaces corresponding to dihedral angle rotations in the middle of the helix have been obtained and compared with the quadratic approximation to those surfaces. The magnitudes and correlations between the fluctuations obtained by averaging over the adiabatic energy surface most closely resemble the vibrational results. Of particular interest is the fact that hydrogen bonds play a relatively small role in the local dihedral angle fluctuations, though the hydrogen bonds are important in the energy of overall length changes.     

A CD study of the α-helix nucleation hypothesis

One of the dilemmas in predicting the secondary structure of proteins from their amino acid propensity for a given conformation is the presence of all amino acids in all types of secondary structure, regardless of their propensity for that specific structure. One explanation is the nucleation hypothesis that only a few residues with a strong propensity for the secondary structure, such as the α-helix structure, initiates its formation and propagates the structure through indifferent sequences until strong breakers terminate the growth on both ends. Eight 15-mer peptides were studied to examine the α-helix nucleation hypothesis. The nucleation sequence of VAEAK, with high helix propensity, was mixed with an indifferent sequence of TSDSR in all possible permutations. From the percent α-helix structure derived from the CD at 222 nm, it appears that helicity does not propagate through the indifferent sequence. © 1994 John Wiley & Sons, Inc.