Miklós Hollósi

Metal ion-induced conformational changes of phosphorylated fragments of human neurofilament (NF-M) protein.

The NF-M subunit of human neurofilaments has a C-terminal repeating 13-mer sequence. The 13-mer (Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly) (NF-M13) and 17-mer (Glu-Glu-Lys-Gly)-(NF-M13) sequences were synthesized, as were both the mono- and diphosphorylated Ser species. Circular dichroism (c.d.) studies and c.d. titrations with Al3+ and Ca2+ were performed. The conformation of the phosphorylated and unphosphorylated material was random in water. Deconvolution of the c.d. spectra, in trifluoroethanol, of the untitrated samples yielded a high content of unordered structure, similar to the poly-L-proline II structure. Titration of the phosphorylated species with Al3+ or Ca2+ caused a surprising conformational change to occur, yielding a high content of beta-pleated sheet structure. A mechanism of metal binding to the phosphofragments is proposed which may be relevant to the formation of neurofibrillary tangles in Alzheimers disease.

Automated solid-phase synthesis of glycopeptides. Incorporation of unprotected mono- and disaccharide units of n-glycoprotein antennae into T cell epitopic peptides

Abstract A new reagent for custom synthesis of N-glycosylated peptides, Fmoc-Asn[GlcNAc(1»4)-GlcNAc]-OH, has been prepared. The O-glycosidic bond survived the trifluoroacetic acid cleavage. High coupling efficiencies were obtained when this glycoamino acid derivative was used for solid-phase synthesis of a test peptide and a T cell epitopic dodecapeptide containing an Asn residue in mid-chain position. An automated peptide synthesizer was used to produce the parent Asn peptide and its N-acetyl-glycosyl mono- and disaccharide derivatives.

Phosphorylation loops in synthetic peptides of the human neurofilament protein middle-sized subunit

Peptides containing 13 and 39 amino acid residues and serine-side-chain-phosphorylated (P) analogues thereof, corresponding to human neurofilament protein middle-sized subunit (NF-M), have been synthesized in order to localize the phosphorylation site of this protein. The secondary structure of the nonphosphorylated peptides, determined by circular dichroism (CD) measurements, predicted secondary structural calculations and energy conformational calculations, was suggested to be a series of alternating type I (III) β-turns and 310 or α-helices. By contrast, the phosphorylated peptides exhibit a unique conformation, probably due to salt bridges between the phosphoserine and the lysine residues. This has provided the first clear evidence that phosphorylation induces conformational changes among these synthetic peptides and presumably, in NF proteins as well. These phosphorylation loops might be the major recognition sites of the neurofilament protein-directed kinases.

Peptide models XXIV: An ab initio study on N-formyl-L-prolinamide with trans peptide bond. The existence or non-existence of αL and ϵL conformations

Abstract N-formyl-L-prolinamide was subjected to geometry optimization at three levels of theory: HF/3-21G, HF/6-31G (d) and B3LYP/6-31G (d). At all three levels of computation the global minimum was γ L (inverse γ -Turn) backbone conformation with two ring-puckered forms “UP” and “DOWN”. At HF/3-21G level of theory three backbone conformations were found γ L , ϵ L , and α L . At higher levels of theory the ϵ L , and α L conformations disappeared. The “UP” and “DOWN” ring-puckered forms, in the γ L backbone conformation, led to practically identical vibrational spectra at the B3LYP/6-31G (d) level of theory.

Fmoc-protected, glycosylated asparagines potentially useful as reagents in the solid-phase synthesis of N-glycopeptides

1-Amino 1-deoxy derivatives of unprotected O-beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-D-glucopyrano se, 2-acetamido-2-deoxy-D-galactose, D-galactose, lactose, D-fucose, D-mannose, and 2-deoxy-D-arabino-hexose were prepared and acylated with N-fluorenylmethoxycarbonylaspartic acid alpha-tert-butyl ester. The anomeric configuration of the N-glycosyl bond (including that of the mannose derivative) in each of the purified compounds was shown to be beta. The probable stability of the N-glycosyl and glycosidic bonds during the conditions of solid-phase peptide synthesis was investigated by treatment of the glycosylated asparagine derivatives with different concentrations of trifluoroacetic acid. Based on their stability, we found that Fmoc-Asn(sugar)-OH derivatives are excellent candidates for automated synthesis of biologically active glycopeptides.

Peptide models XIX: Side-chain conformational energy surface E = ƒ(χ1,χ2) and amide I vibrational frequencies of N-formyl-l-phenylalaninamide (For-Phe-NH2) in its γL or γinv or C7eq backbone conformation

In a study of cross sections of the E = ƒ(χ1,χ2) side-chain conformational potential energy surface of the γL or c7eq backbone conformation of For-l-Phe-NH2, it was found that there are three conformations (g +, a and g −) due to rotation about the Cαχ1 Cβ bond. It should be emphasised that the γL backbone conformation is conserved during rotation about χ1. However, there is only one unique conformation along the rotation about the Cβχ2 Ph bond. The CH2Ph group showed greater stabilisation, with respect to hydrogen (Gly), than the CH3 (Ala) or CH2OH (Ser) substituents. The hydrogen-bonded CO (amide 1) vibrational frequency is split into two bands due to the coupling of the CO stretching and NH2 scissoring modes of motion. The other carbonyl, not involved in hydrogen bonding, has a characteristic single IR band with a relatively high frequency. The orientation of the Ph group has no appreciable effect on these vibrational frequencies.

PEPTIDE MODELS XXII. A CONFORMATIONAL MODEL FOR AROMATIC AMINO ACID RESIDUES IN PROTEINS. A COMPREHENSIVE ANALYSIS OF ALL THE RHF/6-31+G CONFORMERS OF FOR-L-PHE-NH2

Abstract Phenylalanine is expected to have conformational features similar to the other three naturally occurring aromatic amino acid residues: Tyr, Trp and His. Previous ab initio structure determinations resulted in 19 different conformers of HCO–L–Phe–NH2 at the RHF/3–21G level of theory. The present work summarises the results of a comprehensive analysis incorporating RHF/3–21G, RHF/6–31+G*, RHF/6–31+G*//RHF/3–21G and B3LYP/6–311++G**//RHF/3–21G data as some of the previously established minima have vanished with the larger basis set, three out of the 19 stationary points having migrated during the optimisation. On top of that, the conformational building unit of the right-handed helix-like (αL) conformation and that of the polyproline II (eL) conformation are still missing from the E=E(φ,ψ) surface. The optimised ab initio structures are also analysed in the context of –Phe– conformers taken from a large X-ray database on non-homologous proteins incorporating a total of 158 664 amino acid residues.

Solid-phase synthesis of glycopeptides: synthesis of Nα-Flourenylmethoxycarbonyl L-asparagine Nβ-glycosides

Abstract It was found that crude 1 -glycosylamines, prepared with saturated aqueous NH 4 HCO 3 from reducing sugars, can be coupled with Fmoc-Asp(OPfp)-O t Bu in N,N-dimethylformamide-water mixtures. Purification and removal of the t Bu group results in Nβ- glycosides of Fmoc-Asn-OH which can be used for the solid phase synthesis of glycopeptides.

Peptide models XIII. Side-chain conformational energy surface E = E(χ1, χ2) of N-formyl-l-serinamide (For-l-Ser-NH2) in its γL or C7eq backbone conformation

Since each torsional mode (χ1 about the CαCβ and χ2 about the CβO bond) of the side-chain of N-formyl-lserinamide, For-l-Ser-NH2, is expected to have three minima (g+, a, g−), the side chain conformational potential energy surface (PES) E = E(χ1, χ2) is expected to have, in the ideal case, nine legitimate minima. Taking 30 ° intervals along χ1 and χ2 a 12 × 12 grid of points has been generated to examine the side-chain conformational PES using a rigid γL(C7eq) backbone conformation (γL; φ = −75 °, ψ = +75 °). Six out of the nine expected minima have been located on the ab initio PES generated for the γL backbone conformation, at the HF/3-21G level of theory. However, three conformations were missing. The relaxed geometries of the remaining six side-chain conformations have been determined by gradient geometry optimization.

Studies on the conformation of β-endorphin and its constituent fragments in water and trifluoroethanol by CD spectroscopy

Structure-function studies on P-LPH fragments have revealed that the in vitro opiate agonist activity is an exclusive property of fragments containing the complete structure of Met-enkephalin (LPH (61-65)) [ 1 ] at their NH*-terminus and that the biological potency is a function of the chain length of the peptides [2-61. Peptides intermediate between Metenkephalin and LPH (61-9 1) (named fl-endorphin by Li and Chung, [7] ) were found to be less potent than Met-enkephalin in different in vitro bioassays [2-61. Elongation of the peptide chain of LPH (61-79) by addition of the 12 COOH-terminal residues of fl-LPH [4-61 to form P-endorphin caused a dramatic change in the biological properties of the molecule. Among all the fragments studied so far, /3-endorphin is the most active in receptor binding [2,7] in guinea-pig ileum bioassays [3-61 and the least active in mouse vas deferens test [5,6]. In addition, &endorphin exerts the most profound analgesic [2,4,8,9] and behavioral effects [ 10,l l] when administered intraventricularly [2,4,8,10,11] or intravenously [9] to different animals. From the above relationships the impression has