Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Secondary structure”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 991 records · Page 55Linked to original sources
PubMed1996

Secondary structure, membrane localization, and coassembly within phospholipid membranes of synthetic segments derived from the N- and C-termini regions of the ROMK1 K+ channel.

The hydropathy plot of the inwardly rectifying ROMK1 K+ channel, which reveals two transmembrane and a pore region domains, also reveals areas of intermediate hydrophobicity in the N terminus (M0) and in the C terminus (post-M2). Peptides that correspond to M0, post-M2, and a control peptide, pre-M0, were synthesized and characterized for their structure, affinity to phospholipid membranes, organizational state in membranes, and ability to self-assemble and coassemble in the membrane-bound state. CD spectroscopy revealed that both M0 and post-M2 adopt highly alpha-helical structures in 1% SDS and 40% TFE/water, whereas pre-M0 is not alpha-helical in either 1% SDS or 40% TFE/water. Binding experiments with NBD-labeled peptides demonstrated that both M0 and post-M2, but not pre-M0, bind to zwitterionic phospholipid membranes with partition coefficients of 10(3)-10(5) M-1. A surface localization for both post-M2 and M0 was indicated by NBD shift, tryptophan quenching experiments with brominated phospholipids, and enzymatic cleavage. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/ acceptor (rhodamine) peptides revealed that M0 and post-M2 can coassemble in their membrane-bound state, but cannot self-associate when membrane-bound. The results are in agreement with recent data indicating that amino acids in the carboxy terminus of inwardly rectifying K+ channels have a major role in specifying the pore properties of the channels (Taglialatela M, Wible BA, Caporaso R, Brown AM, 1994 Science 264:844-847; Pessia M, Bond CT, Kavanaugh MP, Adelman JP, 1995, Neuron 14:1039-1045). The relevance of the results presented herein to the suggested model for the structure of the ROMK1 channel and to general aspects of molecular recognition between membrane-bound polypeptides are also discussed.

Amino Acid Sequence↗
PubMed1997

Heteronuclear NMR assignments and secondary structure of the coiled coil trimerization domain from cartilage matrix protein in oxidized and reduced forms.

The C-terminal oligomerization domain of chicken cartilage matrix protein is a trimeric coiled coil comprised of three identical 43-residue chains. NMR spectra of the protein show equivalent magnetic environments for each monomer, indicating a parallel coiled coil structure with complete threefold symmetry. Sequence-specific assignments for 1H-, 15N-, and 13C-NMR resonances have been obtained from 2D 1H NOESY and TOCSY spectra, and from 3D HNCA, 15N NOESY-HSQC, and HCCH-TOCSY spectra. A stretch of alpha-helix encompassing five heptad repeats (35 residues) has been identified from intra-chain HN-HN and HN-H alpha NOE connectivities. 3JHNH alpha coupling constants, and chemical shift indices. The alpha-helix begins immediately downstream of inter-chain disulfide bonds between residues Cys 5 and Cys 7, and extends to near the C-terminus of the molecule. The threefold symmetry of the molecule is maintained when the inter-chain disulfide bonds that flank the N-terminus of the coiled coil are reduced. Residues Ile 21 through Glu 36 show conserved chemical shifts and NOE connectivities, as well as strong protection from solvent exchange in the oxidized and reduced forms of the protein. By contrast, residues Ile 10 through Val 17 show pronounced chemical shift differences between the oxidized and reduced protein. Strong chemical exchange NOEs between HN resonances and water indicate solvent exchange on time scales faster than 10 s, and suggests a dynamic fraying of the N-terminus of the coiled coil upon reduction of the disulfide bonds. Possible roles for the disulfide crosslinks of the oligomerization domain in the function of cartilage matrix protein are proposed.

Amino Acid Sequence↗
PubMed2005

Different secondary structure elements as scaffolds for protein folding transition states of two homologous four-helix bundles.

Comparison of the folding processes for homologue proteins can provide valuable information about details in the interactions leading to the formation of the folding transition state. Here the folding kinetics of 18 variants of yACBP and 3 variants of bACBP have been studied by Phi-value analysis. In combination with Phi-values from previous work, detailed insight into the transition states for folding of both yACBP and bACBP has been obtained. Of the 16 sequence positions that have been studied in both yACBP and bACBP, 5 (V12, I/L27, Y73, V77, and L80) have high Phi-values and appear to be important for the transition state formation in both homologues. Y31, A34, and A69 have high Phi-values only in yACBP, while F5, A9, and I74 have high Phi-values only in bACBP. Thus, additional interactions between helices A2 and A4 appear to be important for the transition state of yACBP, whereas additional interactions between helices A1 and A4 appear to be important for the transition state of bACBP. To examine whether these differences could be assigned to different packing of the residues in the native state, a solution structure of yACBP was determined by NMR. Small changes in the packing of the hydrophobic side-chains, which strengthen the interactions between helices A2 and A4, are observed in yACBP relative to bACBP. It is suggested that different structure elements serve as scaffolds for the folding of the 2 ACBP homologues.

Escherichia coli Proteins↗
PubMed1998

Characterization of the secondary structure and membrane interaction of the putative membrane anchor domains of prostaglandin I2 synthase and cytochrome P450 2C1.

Prostaglandin I2 synthase (PGIS) produces prostaglandin I2 (PGI2) which has opposite actions on platelet aggregatory and vasoconstrictive properties compared to thromboxane A2 (TXA2) produced from the same substrate by another P450 enzyme, thromboxane A2 synthase (TXAS). PGIS and TXAS have only 16% amino acid sequence identity. Hydropathy analysis suggests that the putative NH2-terminal membrane anchor domain of PGIS is similar to many other membrane-bound microsomal P450s, which are believed to be anchored by a single transmembrane segment, and thus different from the TXAS anchor, which appears to have two transmembrane segments. To characterize the membrane anchor function of the PGIS NH2-terminal region, we have used the peptidoliposome reconstitution assay to identify the membrane anchor segment in the PGIS NH2-terminal domain and compared it with the anchor segment of P450 2C1. Four peptides, mimicking putative NH2-terminal membrane anchor segments of PGIS and P450 2C1, containing residues 1-28 (PGIS-LP1 and P450 2C1-LP1) or residues 25-54 (PGIS-LP2 and P450 2C1-LP2), were synthesized and their ability to insert in a lipid bilayer was evaluated. The results indicated that both LP1 peptides of PGIS and P450 2C1 became bound to the lipid bilayer, whereas both LP2 peptides did not bind the lipid. The two LP1 peptides were further characterized as to their conformation using CD spectroscopy. Helical structure induced in these peptides by addition of trifluoroethanol, dodecylphosphocholine, or incorporation into liposomes indicated that these segments tend to adopt a helical structure in a hydrophobic environment and thus could function as membrane anchor segments. These results support the hypothesis that PGIS and TXAS interact with the endoplasmic reticulum membrane in different ways, in which the NH2-terminal anchor domain of PGIS, as with P450 2C1, appears to have a single transmembrane segment.

Amino Acid Sequence↗
PubMed1995

The secondary structure of phospholamban: a two-dimensional NMR study.

Phospholamban (PLN) is an intrinsic membrane protein of 52 amino acids which regulates the Ca2+ pump of the sarcoplasmic reticulum of heart, slow-twitch and smooth muscle (SR): it is normally assumed to exist in the membrane as a homopentamer. A monomeric analogue of phospholamban PLN(C41F), in which Cys41 was replaced by a Phe, was synthesized and its conformation studied by 1H NMR spectroscopy in a 1:1 mixture of chloroform/methanol. Most of the resonances in the 1H NMR spectra were assigned. The work has shown that the C-terminal hydrophobic portion forms a very stable alpha-helix. The hydrophilic N-terminal part adopts an alpha-helix configuration which is much less stable except for the stretch containing the phosphorylation sites.

Amino Acid Sequence↗
PubMed2000

Glycosaminoglycan-binding properties and secondary structure of the C-terminus of netrin-1.

Netrins are soluble neurite-outgrowth-promoting proteins related to the laminin B2 chain. Since these proteins and their receptor DCC (the "deleted in colorectal carcinoma" gene product) bind heparin, glycosaminoglycans may modulate their biological actions in a similar fashion as described for several other ligand-receptor systems. Here we show that a polypeptide encompassing the C-terminal cluster of basic amino acids of netrin-1 (i) adopts an alpha-helical conformation in water-trifluoroethanol mixtures according to circular dichroism experiments and (ii) binds electrostatically to heparin with high affinity under physiological ionic conditions (K(D) = 15 nM for the binding to immobilized heparin according to surface plasmon resonance, K(D) = 50 nM in solution as determined with isothermal titration calorimetry). These data indicate that the cluster of basic amino acids at the C-terminus of netrin-1 forms an alpha-helical structural element which can contribute to the glycosaminoglycan-binding activity of this neurotrophic guidance molecule.

Amino Acid Sequence↗
PubMed1995

Free energy determinants of secondary structure formation: I. alpha-Helices.

The Zimm-Bragg parameters s and sigma are calculated for the helix-coil transition of poly-L-alanine. The theoretical approach involves evaluating gas phase conformational energies for both coil and helical states using the CHARMM potential function and accounting for solvation effects with various continuum solvation models. Conformational free energies are then incorporated into a formalism developed by Go et al. for the calculation of s and sigma. Calculated values for both s and sigma as well as the enthalpy change associated with helix formation are in good agreement with experimental data when the Finite Difference Poisson-Boltzmann (FDPB) method is used to treat solvent effects. The driving force for the helix-coil transition is analyzed in terms of individual free energy components. Hydrogen bond formation is found to contribute little to helix stability because the internal hydrogen bonding energy is largely canceled by the large free energy cost associated with removing polar groups from water. The entropic cost associated with fixing backbone dihedral angles in the helical conformation is found to be approximately 7 e.u./residue (about 2 kcal/mol at room temperature). The major driving force favoring helix formation can be associated with interactions including enhanced van der Waals interactions in the close-packed helix conformation and the hydrophobic effect. These contribute about 2 kcal/mol favoring the helical state. The differences in helical propensities between alanine and glycine are attributed primarily to hydrophobic and packing interactions involving the C beta with a smaller contribution arising from increased conformational freedom for glycine in the coil state. The description of helix formation presented here is consistent with previous conclusions regarding tertiary structure formation which suggest that hydrophobic and close-packed interactions provide stability while hydrogen bond formation constitutes a structural constraint imposed by the high free energy cost associated with burying unsatisfied hydrogen bonding groups. alpha-Helix formation may thus be viewed as a form of hydrophobic collapse constrained by the requirement that polar groups be either exposed to solvent or form hydrogen bonds. More generally it appears from this study that for a folding model to be a realistic, it must properly account for the chemical nature of the polypeptide chain, particularly the solvation energetics of amide groups.

Amino Acid Sequence↗
PubMed1995

Free energy determinants of secondary structure formation: II. Antiparallel beta-sheets.

The factors that determine the stability of antiparallel beta-sheets are considered via a theoretical analysis of conformational free energies. A series of idealized model polyalanine beta-sheets are built with constraints such that the angular geometry of hydrogen bonding varies in the range observed in proteins while hydrogen bonding distance remains fixed. The conformations of the sheets generated in this way have a broad distribution of twist angles ranging from highly twisted left-handed to highly twisted right-handed orientations. The association free energies of the sheets are calculated with a gas phase CHARMM potential and FDPB/gamma solvation models. Left-handed structures are found to be less stable than right handed structures due to intrachain steric hindrance in isolated left-handed strands. This explains why antiparallel beta-sheets in proteins are invariably twisted in the right-handed direction. The free energy surface for right-handed sheets shows particular preference for conformations ranging from flat to those that exhibit a pronounced right-handed twist. This suggests that antiparallel beta-sheets can adopt a variety of right-handed conformations, a result that is consistent with observations on known proteins. In parallel with our study of alpha-helices we find that van der Waals and hydrophobic interactions are the primary factor stabilizing polyalanine beta-sheets, while electrostatic interactions including hydrogen bonding are found to be destabilizing. However, in contrast to helices, the net change in conformational free energy involving only backbone-backbone interactions (including beta-carbons) is not sufficient to overcome the loss in configurational entropy that accompanies sheet formation. Rather we suggest that cross-strand non-polar side-chain-side-chain interactions are essential for sheet formation, explaining why large non-polar amino acids have the greatest sheet forming propensities. Thus, sheet propensities involve pairwise interactions and are expected to be context dependent, as has been observed in recent experiments.

Amino Acid Sequence↗
PubMed1996

Use of a conformationally restricted secondary structural element to display peptide libraries: a two-stranded alpha-helical coiled-coil stabilized by lactam bridges.

A model for an alpha-helical peptide library based on a lactam bridged stabilized two-stranded alpha-helical coiled-coil is described. Sites for library display were incorporated in the middle of the peptide sequence of the most solvent accessible sites of a coiled-coil. A comparison was made between this coiled coil and a native coiled-coil based on the same sequence but lacking the lactam bridges. A lactam bridged peptide where the hydrophobic repeat consisted of all alanine residues, such that the tendency to dimerize would be diminished, was also prepared. This enabled us to determine the role tertiary interactions play in maintaining library positions in a helical conformation. The consensus sequence derived from a Zn finger library screened against an IgA reactive against the lipopolysaccharide of Shigella flexneri was transplanted into the peptides. CD spectroscopy revealed that although both coiled-coils are highly helical at 100 microM, the lactam bridge enhanced dimerization and allow the peptide to maintain its coiled-coil conformation at lower peptide concentrations. The helical content of the alanine based peptide was 69% and was independent of concentration over a range of 1.4 to 1410 microM. Urea denaturation studies indicated that the coiled-coils were considerably more stable than the alanine based peptide and that the lactam bridge coiled-coil was more stable than the native coiled coil by 1.6 kcal/mol. The lactam bridged coiled-coil was found to inhibit binding of the Zn finger peptide to the IgA in a concentration dependent manner with an IC50 of 5.0 microM whereas the peptide lacking the lactam bridges was much less effective in inhibiting binding. The alanine peptide was less active than the lactam bridged coiled-coil but more effective than the native coiled-coil with an IC50 of 16 microM. The versatility of the lactam stabilized coiled-coil template was demonstrated by incorporating five Gly residues into the library display sites. While the native coiled-coil adopted a random conformation the lactam bridged coiled-coil was 59% helical. Incorporation of a Cys-Gly-Gly linker to the N terminus and formation of a disulfide bond stabilized the peptide to the extent that it adopted a highly helical coiled-coil (94%) with a [urea]1/2 value of 5.9 M. Since the five glycine residues represent one of the most destabilizing combinations of amino acids that would be encountered at the five library display sites (with the exception of Pro), this stabilized coiled-coil should maintain its folded conformation regardless of the amino acids occupying the library positions.

Amino Acid Sequence↗
PubMed1999

Effects of varying the local propensity to form secondary structure on the stability and folding kinetics of a rapid folding mixed alpha/beta protein: characterization of a truncation mutant of the N-terminal domain of the ribosomal protein L9.

The N-terminal domain of the ribosomal protein L9 forms a split betaalphabeta structure with a long C-terminal helix. The folding transitions of a 56 residue version of this protein have previously been characterized, here we report the results of a study of a truncation mutant corresponding to residues 1-51. The 51 residue protein adopts the same fold as the 56 residue protein as judged by CD and two-dimensional NMR, but it is less stable as judged by chemical and thermal denaturation experiments. Studies with synthetic peptides demonstrate that the C-terminal helix of the 51 residue version has very little propensity to fold in isolation in contrast to the C-terminal helix of the 56 residue variant. The folding rates of the two proteins, as measured by stopped-flow fluorescence, are essentially identical, indicating that formation of local structure in the C-terminal helix is not involved in the rate-limiting step of folding.

Chi-Square Distribution↗
PubMed1998

Porcine cerebroside sulfate activator (saposin B) secondary structure: CD, FTIR, and NMR studies.

Cerebroside sulfate activator protein (CSAct or saposin B) is one of a group of heat stable, low-molecular-weight proteins that appear to share a common structural motif. These have been referred to as saposin-like proteins and are thought to share a multiple amphipathic helical barrel structure with a conserved pattern of disulfide linkages. Porcine kidney CSAct was prepared in high purity and consisted of three major glycosylated subforms. The protein was studied by physical-chemical methods and evaluated by various methods for structural prediction. All suggest that CSAct has high amounts of alpha-helical conformation and little if any beta-sheet. Circular dichroism (CD) studies indicate 45-50% helical conformation depending on buffer and temperature. There was only a moderate loss in helical content with increasing temperature and no indication of thermal denaturation. Fourier transform infrared spectroscopy (FTIR) measurements on deuterium hydrated self-films also indicated a predominantly helical structure. Helical axis orientation was investigated by both oriented CD and FTIR dichroism, which suggested that the helical axes were roughly parallel and oriented along the axis of the surface on which the self-films had been deposited. One-dimensional nuclear magnetic resonance spectra showed large chemical shift dispersion, indicating a defined tertiary structure with little variation between 6 and 85 degrees C. NOESY spectra failed to show the strong NOE cross peaks expected for a highly helical conformation. This may indicate short-term conformational flexibility within the helices or molecular aggregation at the high protein concentrations employed. These observations are consistent with the 3-4-helix bundle motif suggested for saposin-like proteins by various predictive algorithms.

Algorithms↗
PubMed1994

1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin.

Sequence-specific backbone 1H and 15N resonance assignments have been made for 95% of the amino acids in sperm whale myoglobin, complexed with carbon monoxide (MbCO). Many assignments for side-chain resonances have also been obtained. Assignments were made by analysis of an extensive series of homonuclear 2D spectra, measured with unlabeled protein, and both 2D and 3D 1H-15N-correlated spectra obtained from uniformly 15N-labeled myoglobin. Patterns of medium-range NOE connectivities indicate the presence of eight helices in positions that are very similar to those found in the crystal structures of sperm whale myoglobin. The resonance assignments of MbCO form the basis for determination of the solution structure and for hydrogen-exchange measurements to probe the stability and folding pathways of myoglobin. They will also form a basis for assignment of the spectra of single-site mutants with altered ligand-binding properties.

Amino Acid Sequence↗
PubMed1994

1H, 15N and 13C resonance assignments, secondary structure, and the conformation of substrate in the binary folate complex of Escherichia coli dihydrofolate reductase.

By using fully 15N- and 15N/13C-labeled Escherichia coli dihydrofolate reductase, the sequence-specific 1H and 15N NMR assignments were achieved for 95% of the backbone resonances and for 90% of the 13C alpha resonances in the binary folate complex. These assignments were made through a variety of three-dimensional proton-detected 15N and 13C experiments. A smaller but significant subset of side-chain 1H and 13C assignments were also determined. In this complex, only one 15N or 13C resonance was detected per 15N or 13C protein nucleus, which indicated a single conformation. Proton-detected 13C experiments were also performed with unlabeled DHFR, complexed with 13C-7/13C-9 folate to probe for multiple conformations of the substrate in its binary complex. As was found for the protein resonances, only a single bound resonance corresponding to a productive conformation could be detected for C-7. These results are consistent with an earlier report based on 1H NMR data [Falzone, C.J. et al. (1990) Biochemistry, 29, 9667-9677] and suggest that the E. coli enzyme is not involved in any catalytically unproductive binding modes in the binary complex. This feature of the E. coli enzyme seems to be unique among the bacterial forms of DHFR that have been studied to date.

Amino Acid Sequence↗
PubMed1993

13C alpha-NMR assignments of melittin in methanol and chemical shift correlations with secondary structure.

Melittin is a naturally occurring hexacosa peptide which forms an amphiphilic helix in methanol, a random coil in water, and a tetramer of helices at basic pH or in the presence of a high salt concentration. The monomeric structure in methanol has been well characterized by proton NMR (Pastore et al. (1989) Eur. Biophys. J., 16, 363-367). In the present paper, chemical shifts of the backbone alpha-carbons of melittin in methanol were determined by mapping previously published alpha-proton shifts (Bazzo et al. (1988) Eur. J. Biochem., 173, 139-146), to natural abundance (1H)13C cross peaks appearing in the 2D heteronuclear multiple-quantum NMR spectrum. Changes in chemical shifts consequent to stepwise increases in the percentage of water in a mixed methanol/water solvent system were observed in similar spectra. The alpha-carbon shifts varied more smoothly than the corresponding alpha-proton shifts and were found to correlate with the transition from the helix to the random coil conformer in parallel with changes in the circular dichroism spectrum. Chemical shifts of this peptide are interpreted with regard to the current database of assignments in proteins of known 3D structure (Wishart et al. (1991) J. Mol. Biol., 222, 311-333). The N-terminal region of the peptide shows increased flexibility at lower methanol concentrations, as evidenced by the merger of the alpha-proton resonances of G1 (at 40% and 15% methanol) and G3 (at 15% methanol). Conformational exchange rates for G1 and G3 were estimated by comparison of the experimental spectra with simulated spectra and found to be as large as 4000 s-1 for G1 in 40% and 15% methanol and 600 s-1 for G3 in 15% methanol. Overall, these 1H and 13C chemical shift data support the description of monomeric melittin in methanol currently evolving in the literature and suggest a structure composed of a linked pair of helices with different structural stabilities, each of which experiences dynamical fraying at its free terminus.

Calcium Isotopes↗
PubMed1994

The effect of vesical outlet obstruction on the protein secondary structure of the mucosa and serosa in rabbit bladder wall.

The biochemical composition of physiologically moist mucosa and serosa of rabbit bladder before and after bladder outlet obstruction was determined by means of FT-IR spectroscopy with the ATR method and second-derivative analysis. A predominantly beta-sheet structure was found in the amide I band for mucosa and serosa before and after obstruction, but the random coil structure increased in both obstructed bladder samples. However, the major beta-sheet structure associated with some alpha-helical structure in the amide II band of mucosa and serosa for non-obstructed bladder changed into a predominantly alpha-helical structure after bladder obstruction. The obstructed bladder serosa was more pronounced. The amount of glycoproteins doubled in the obstructed bladder serosa, but did not change in the bladder mucosa.

Animals↗
PubMed1993

Secondary structural changes of large and small fragments of bovine serum albumin in thermal denaturation and in sodium dodecyl sulfate denaturation.

The helicities in various fragments of bovine serum albumin (BSA) were examined in the thermal denaturation and in sodium dodecyl sulfate (SDS) denaturation. The thermal denaturation was examined in a temperature range between 2 and 65 degrees C. The helicity decreased with a rise of temperature and it recovered to some degree upon cooling temperature. A rather high reversibility was observed in the BSA fragments, which were located in the N-terminal of the parent protein and then contained the first large loop with no disulfide bridge. The high reversibility was available also for the helicity in the first large loop of the fragment, disulfide bridges of which were reduced. The fragments, which were smaller than one domain, became unstable in the SDS denaturation. The helicities of such fragments decreased in lower SDS concentrations compared with those of the intact BSA and the large fragments, which contained one or more domains. A resistance to the SDS denaturation appeared in the helices of every large loop even after the fragmentation. On the other hand, helicities of the fragments decreased to 20-25% upon the reduction of disulfide bridges. However, the helicities of these fragments increased to 35-40% in the SDS denaturation.

Animals↗
PubMed1996

Prediction of protein secondary structure from amino acid sequence.

The conformational parameters Pk for each amino acid species (j = 1-20) of sequential peptides in proteins are presented as the product of P(i,k), where i is the number of the sequential residues in the kth conformational state (k = alpha-helix, beta-sheet, beta-turn, or unordered structure). Since the average parameter for an n-residue segment is related to the average probability of finding the segment in the kth state, it becomes a geometric mean of (Pk)av = II (P(i,k))1/n with amino acid residue i increasing from 1 to n. We then used ln(Pk)av to convert a multiplicative process to a summation, i.e., ln(Pk)av = (1/n)sigma P(i,k) (i = 1 to n) for ease of operation. However, this is unlike the popular Chou-Fasman algorithm, which has the flaw of using the arithmetic mean for relative probabilities. The Chou-Fasman algorithm happens to be close to our calculations in many cases mainly because the difference between their Pk and our ln Pk is nearly constant for about one-half of the 20 amino acids. When stronger conformation formers and breakers exist, the difference become larger and the prediction at the N- and C-terminal alpha-helix or beta-sheet could differ. If the average conformational parameters of the overlapping segments of any two states are too close for a unique solution, our calculations could lead to a different prediction.

Algorithms↗