Turn Secondary Structure Research Articles

Benzotriazepine synthesis, conformational analysis, and biological properties

Various benzotriazepine ring systems have been synthesized and examined for biological activity. Moreover, the 1,3,4-benzotriazepin-2-one has been shown to act as a constrained amino amide surrogate in mimics of peptide turn secondary structures. This review examines syntheses of the different benzotriazepine ring systems and heterocycle fused variants as well as their conformational and biological properties.

An amino acid code for irregular and mixed protein packing.

To advance our understanding of protein tertiary structure, the development of the knob-socket model is completed in an analysis of the packing in irregular coil and turn secondary structure packing as well as between mixed secondary structure. The knob-socket model simplifies packing based on repeated patterns of two motifs: a three-residue socket for packing within secondary (2°) structure and a four-residue knob-socket for tertiary (3°) packing. For coil and turn secondary structure, knob-sockets allow identification of a correlation between amino acid composition and tertiary arrangements in space. Coil contributes almost as much as α-helices to tertiary packing. In irregular sockets, Gly, Pro, Asp, and Ser are favored, while in irregular knobs, the preference order is Arg, Asp, Pro, Asn, Thr, Leu, and Gly. Cys, His,Met, and Trp are not favored in either. In mixed packing, the knob amino acid preferences are a function of the socket that they are packing into, whereas the amino acid composition of the sockets does not depend on the secondary structure of the knob. A unique motif of a coil knob with an XYZ β-sheet socket may potentially function to inhibit β-sheet extension. In addition, analysis of the preferred crossing angles for strands within a β-sheet and mixed α-helice/β-sheet identifies canonical packing patterns useful in protein design. Lastly, the knob-socket model abstracts the complexity of protein tertiary structure into an intuitive packing surface topology map.

Prediction of glycoprotein secondary structure using ATR-FTIR

Glycoproteins are important biomolecules with a diverse array of structural and signaling functions in biology. Determination of glycoprotein secondary structure is becoming increasingly important in aiding the understanding of how these molecules function in biological environments and disease. Furthermore, glycoproteins such as mucins are being evaluated in various nano-engineering processes that require knowledge of how the underlying secondary structure might alter in different target environments. We have developed an analytical procedure for predicting the secondary structures of glycoprotein using ATR-FTIR on dry film. Using Bovine submaxillary mucin (BSM) as a glycoprotein model, we determined the additive infrared spectral pattern of acetyl amino sugars and amino acids that could contribute to the absorbance in the Amide I band of BSM through empirical data. We show through subtraction of these spectra how the absorbance pattern of the protein backbone can be determined in order to predict glycoprotein secondary structure. The analysis predicted a predominant pattern of random coil, beta sheet and beta turn secondary structure for BSM after carbohydrate and amino acid spectral subtraction in agreement with other methods. Our relatively simple approach can be applied to predict secondary structure in other glycoproteins.

α N–O turn induced by fluorinated α-aminoxy diamide: synthesis and conformational studies

Two α-aminoxy diamides with fluorinated side chains were synthesized. Their secondary structures characterization was carried out by 1H NMR, and IR spectrometries as well as X-ray crystallography studies. α N–O turn secondary structures are adopted insusceptibly by side-chain-fluorinated α-aminoxy residues. Thus the fluorinated α-aminoxy diamide can be a potential residue as a biological tracer to be incorporated into aminoxy peptides.

A Logical OR Redundancy within the Asx-Pro-Asx-Gly Type I β-Turn Motif

Turn secondary structure is essential to the formation of globular protein architecture. Turn structures are, however, much more complex than either α-helix or β-sheet, and the thermodynamics and folding kinetics are poorly understood. Type I β-turns are the most common type of reverse turn, and they exhibit a statistical consensus sequence of Asx-Pro-Asx-Gly (where Asx is Asp or Asn). A comprehensive series of individual and combined Asx mutations has been constructed within three separate type I 3:5 G1 bulge β-turns in human fibroblast growth factor-1, and their effects on structure, stability, and folding have been determined. The results show a fundamental logical OR relationship between the Asx residues in the motif, involving H-bond interactions with main-chain amides within the turn. These interactions can be modulated by additional interactions with residues adjacent to the turn at positions i+4 and i+6. The results show that the Asx residues in the turn motif make a substantial contribution to the overall stability of the protein, and the Asx logical OR relationship defines a redundant system that can compensate for deleterious point mutations. The results also show that the stability of the turn is unlikely to be the prime determinant of formation of turn structure in the folding transition state.

Understanding the mechanism of β‐sheet folding from a chemical and biological perspective

Perturbing the structure of the Pin1 WW domain, a 34-residue protein comprised of three beta-strands and two intervening loops has provided significant insight into the structural and energetic basis of beta-sheet folding. We will review our current perspective on how structure acquisition is influenced by the sequence, which determines local conformational propensities and mediates the hydrophobic effect, hydrogen bonding, and analogous intramolecular interactions. We have utilized both traditional site-directed mutagenesis and backbone mutagenesis approaches to alter the primary structure of this beta-sheet protein. Traditional site-directed mutagenesis experiments are excellent for altering side-chain structure, whereas amide-to-ester backbone mutagenesis experiments modify backbone-backbone hydrogen bonding capacity. The transition state structure associated with the folding of the Pin1 WW domain features a partially H-bonded, near-native reverse turn secondary structure in loop 1 that has little influence on thermodynamic stability. The thermodynamic stability of the Pin1 WW domain is largely determined by the formation of a small hydrophobic core and by the formation of desolvated backbone-backbone H-bonds enveloped by this hydrophobic core. Loop 1 engineering to the consensus five-residue beta-bulge-turn found in most WW domains or a four-residue beta-turn found in most beta-hairpins accelerates folding substantially relative to the six-residue turn found in the wild type Pin1 WW domain. Furthermore, the more efficient five- and four-residue reverse turns now contribute to the stability of the three-stranded beta-sheet. These insights have allowed the design of Pin1 WW domains that fold at rates that approach the theoretical speed limit of folding.

Catalysis with Phosphine-Containing Amino Acids in Various “Turn” Motifs

We have been actively involved in the development of parallel approaches for the discovery of phosphine ligands. Our approach has been based on the incorporation of phosphine-containing amino acids into peptide sequences that are designed to have stable secondary structures. We have examined helical and turn secondary structures and have reported that alkylation of cyclopentenyl acetate with dimethylmalonate can be catalyzed in high enantiomeric excess (ee) with a beta-turn-based ligand. The importance of the peptide secondary structure was demonstrated through the synthesis of a series of peptide ligands where the nature of the turn-forming residues was probed. Additionally, other turn-forming units and a variety of different phosphine-containing amino acids have been examined for their ability to control the selectivity of the allylation reaction. This paper reports the results obtained through the examination of different turn motifs as well as different phosphine substitutions on the "best" turn sequence, Pps-Pro-d-Xxx-Pps.

Aza-amino acid scan for rapid identification of secondary structure based on the application of N-Boc-aza(1)-dipeptides in peptide synthesis.

Azapeptides, peptide analogues in which the alpha-carbon of one or more of the amino acid residues is replaced with a nitrogen atom, exhibit propensity for adopting beta-turn conformations. A general protocol for the synthesis of azapeptides without racemization on solid phase has now been developed by introducing the aza-amino acid residue as an N-Boc-aza(1)-dipeptide. This approach has been validated by the synthesis of six N-Boc-aza(1)-dipeptides and their subsequent introduction into analogues of the C-terminal peptide fragment of the human calcitonin gene-related peptide (hCGRP). By performing an aza-amino acid scan of such antagonist peptides, a set of aza-hCGRP analogues was synthesized to examine the relationship between turn secondary structure and biological activity.

A novel mutation (G233D) in the glycogen phosphorylase gene in a patient with hepatic glycogen storage disease and residual enzyme activity

We identified a novel mutation in the glycogen phosphorylase gene ( PGYL) in a Chinese patient with glycogen storage disease (GSD) type VI. The patient presented with gross hepatomegaly since the age of two without history of any hypoglycemic attack. Otherwise, he was largely asymptomatic. Liver tissue enzyme assays revealed a mild deficiency of total glycogen phosphorylase. Both PGYL and PHKA2 genes were sequenced. The patient was homozygous of a missense mutation G233D in PGYL. This location forms a hairpin turn secondary structure and the small glycine residue is completely conserved in all the orthologous proteins from Escherichia coli to mammals. This is the sixth reported mutation of this form of GSD.

Bend ribbon-forming tetrahydrofuran amino acids.

Short oligomeric chains of C-glycosyl beta-D-arabinofuranose configured tetrahydrofuran amino acids (where the C-2 and C-5 substituents of the tetrahydrofuran ring are cis to each other) exhibit a well-defined repeating turn secondary structure stabilised by (i, i - 2) inter-residue hydrogen bonds. This is in contrast to the epimeric alpha-D-arabinofuranose oligomer (where the C-2 and C-5 substituents of the tetrahydrofuran ring are trans to each other) in which there is no indication of any secondary structure in solution.

Applications of the Mitsunobu reaction in peptide chemistry.

The Mitsunobu reaction--the nucleophilic substitution of an alcoholic hydroxyl group mediated by the redox system trialkylphosphine/dialkyl azodicarobxylate--is widely used in the chemistry of biologically active compounds. The paper deals with applications of the Mitsunobu reaction in amino acid and peptide chemistry. The process provides easy access to many unnatural amino acids and derivatives. Since the reaction occurs with complete inversion of the configuration at the carbinol chiral centre, it can be used for the synthesis of diastereoisomers of hydroxy- and tioprolines. Cyclization of beta-hydroxy amino acid containing peptides under Mitsunobu reaction conditions leads to a constrained peptide that mimics the stabilizing reverse turn secondary structure.

Dissection of the extracellular human interferon gamma receptor alpha-chain into two immunoglobulin-like domains. Production in an Escherichia coli thioredoxin gene fusion expression system and recognition by neutralizing antibodies.

The extracellular interferon gamma receptor alpha-chain (IFN gamma R) is believed to comprise two discrete approximately 110 amino acid immunoglobulin-like domains, perhaps similar to those seen in the crystal structure of the extracellular human growth hormone receptor [De Vos, A. M., Ultsch, M., & Kossiakoff, A. (1992) Science 255, 306-312], a distant relative in the cytokine receptor superfamily. In accord with this idea, we show that these IFN gamma R immunoglobulin-like domains can be produced separately in a soluble form with a native-like fold. The N-terminal domain (residues 1-108), with a Cys105 to Ser105 mutation, was produced at a high level, in a soluble form, as a thioredoxin-interferon gamma receptor fragment fusion protein in the cytoplasm of Escherichia coli. Upon extraction, the receptor Cys60-Cys68 disulfide bond formed spontaneously, to generate a native-like structure directly without the need for refolding. Cleavage of the fusion protein by enterokinase released the receptor fragment (approximately 12 kDa), which was recognized by several neutralizing antibodies with affinities, measured using surface plasmon resonance technology, that were essentially indistinguishable from those seen with the full length extracellular IFN gamma R produced in eukaryotic cells. Circular dichroism and 1D 1H nuclear magnetic resonance spectra indicated that the receptor fragment adopts a folded state, with mainly beta-sheet and reverse turn secondary structure. The second membrane-proximal Ig-like domain of the IFN gamma R (residues 90-229) was produced, albeit less efficiently, and characterized in a similar way. The production of these two independently folded proteins provides experimental support for the two domain organization of the IFN gamma R and opens new avenues for structural studies on these Ig-like molecules by NMR and crystallographic methods.

Isolation, primary structures and metal binding properties of neuronal growth inhibitory factor (GIF) from bovine and equine brain

Human neuronal growth inhibitory factor (GIF) impairs the survival of cultured neurons and is deficient in the brains of Alzheimer's disease victims. We have isolated and sequenced analogous proteins from bovine and equine brain. By comparing their primary structures with those of human, mouse and rat GIFs, a consensus GIF sequence was obtained. Although this exhibits ca. 65% similarity with primary structures of mammalian metallothioneins (MTs), some significant differences are expected in the content of helix and turn secondary structures. In contrast to MTs, which usually bind 7 Zn(II) ions, human, bovine and equine GIFs contain 1–4 Cu(I) and 3–5 Zn(II) ions in species-specific ratios. The observed Cu(I) phosphorescence (λ max 550–590 nm; τ, 100 μs at 77 K) indicates the presence of the cuprous ion. Both bovine Cu 1Cd 5- and the equine Cu 3Cd 3-GIF derivatives (Cd replacing Zn) exhibit cadmium-dependent absorption and CD features between 220–260 nm characteristic of Cd-thiolate clusters similar to those in Cd-MTs.

Analysis of the Steroid Binding Domain of Rat Androgen-Binding Protein*

The site-directed photoaffinity ligand [3H]17 beta-hydroxy-4,6-androstadien-3-one (delta 6-testosterone) was used to label the steroid binding domain of rat androgen-binding protein (rABP). After digestion with trypsin, the major radiolabeled peptide was isolated by reverse phase chromatography. The peptide was found to have the following amino acid sequence: Ile Ala Leu Gly Gly Leu Leu Leu Pro Thr Ser. Gaps in the sequence that one would anticipate if delta 6-testosterone formed an adduct with a single amino acid were not encountered. Several different amino acids appear to have been labeled as expected given the free radical nature of photoactivated delta 6-testosterone. The sequence obtained corresponded to a tryptic peptide (amino acids 171-181) of the rABP precursor. The only other protein having this amino acid sequence was human sex hormone binding globulin. The binding domain lies in a hydrophobic pocket that contains a predicted beta-sheet and turn secondary structure, as would be anticipated given the hydrophobic nature of the steroid molecule. A hydropathy and secondary structure analysis of rABP was performed as a basis for discussing the results of the current study in relation to previous studies on the steroid binding domain on human sex hormone binding globulin.