Krystyna Stachowiak

Mechanism of fluorescence quenching of tyrosine derivatives by amide group

Abstract The difference between fluorescence lifetimes of the following amino acids: phenylalanine (Phe), tyrosine (Tyr), ( O -methyl)tyrosine (Tyr(Me)), (3-hydroxy)tyrosine (Dopa), (3,4-dimethoxy)phenylalanine (Dopa(Me) 2 ) and their amides was used to testify the mechanism of fluorescence quenching of aromatic amino acids by the amide group. On the basis of the Marcus theory of photoinduced electron transfer parabolic relationships between ln k ET and ionization potentials reduced by energy of excitation ( IP −E ∗ 0,0 ) for the above-mentioned amino acids were obtained. This finding indicates the occurrence of photoinduced electron transfer from the excited chromophore group to the amide group.

The role of the Val57 amino-acid residue in the hinge loop of the human cystatin C. Conformational studies of the beta2-L1-beta3 segments of wild-type human cystatin C and its mutants.

Human cystatin C (HCC) is one of the amyloidogenic proteins to be shown to oligomerize via a three‐dimensional domain swapping mechanism. This process precedes the formation of a stable dimer and proceeds particularly easily in the case of the L68Q mutant. According to the proposed mechanism, dimerization of the HCC precedes conformational changes within the β2 and β3 strands. In this article, we present conformational studies, using circular dichroism and MD methods, of the β2‐L1‐β3 (His43‐Thr72) fragment of the HCC involved in HCC dimer formation. We also carried out studies of the β2‐L1‐β3 peptide, in which the Val57 residue was replaced by residues promoting β‐turn structure formation (Asp, Asn, or Pro). The present study established that point mutation could modify the structure of the L1 loop in the β‐hairpin peptide. Our results showed that the L1 loop in the peptide excised from human cystatin C is broader than that in cystatin C. In the HCC protein, broadening of the L1 loop together with the unfavorable L68Q mutation in the hydrophobic pocket could be a force sufficient to cause the partial unfolding and then the opening of HCC or its L68Q mutant structure for further dimerization. We presume further that the Asp57 and Asn57 mutations in the L1 loop of HCC could stabilize the closed form of HCC, whereas the Pro57 mutation could lead to the opening of the HCC structure and then to dimer/oligomer formation.

Photophysics of phenylalanine analogues. Part 1. Constrained analogues of phenylalanine modified at phenyl ring

Abstract Photophysics of constrained analogues of phenylalanine (Phe) modified at phenyl ring: tetrahydroisoquinoline-3-carboxylic acid (Tic), (5-methyl) tetrahydroisoquinoline-3-carboxylic acid ((5-Me)Tic), (2′-methyl)phenylalanine ((2′-Me)Phe) and (2′,6′-dimethyl)phenylalanine ((2′,6′-Me 2 ) Phe) and their simple derivatives obtained by a modification of amino group (acetylation) or carboxylic group (amidation), or by both modifications at the same time was the subject of our investigations. The synthesized compounds were used to study the influence of (i) sterical constraints (cyclization or incorporation of methyl substituent(s) into phenyl ring), (ii) the N-acetylation of the amino group and (iii) the transformation of the carboxylic group into the amide on the photophysical properties of the phenylalanine analogue.

Photophysics of phenylalanine analogues. Part 2. Linear analogues of phenylalanine

Abstract The photophysical properties (fluorescence quantum yields and lifetimes) of linear analogues of phenylalanine (Phe) [β-homophenylalanine (β-Hph), β-phenylalanine (β-Phe), phenyglycine (Phg)] in water at pH=6 and 1 have been measured. The obtained results indicate that relative space location of the amino, carboxyl groups and phenyl chromophore influence on photophysical properties at both pH. In acidic solution (pH=1) lower fluorescence quantum yields and fluorescence lifetimes than observed at pH=6 for all studied compounds suggest that the protonated carboxylic group efficiently quenches phenyl fluorescence. It was found that the proximity of the protonated amino group and the phenyl ring modify the photophysical properties of phenylalanine analogues β-Phe and Phg.

The photophysics of β-homo-tyrosine and its simple derivatives

Synthesis and photophysical studies of (O-methyl)-β-tyrosine (β-tyrosine; an analogue of tyrosine, in which the amino group is moved from the α- to the β-carbon, closer to the phenol ring) and its derivatives with a blocked amino and/or carboxyl group were performed to explain the nature of the fluorescence of tyrosine derived analogues. All β-tyrosine derivatives, except Ac-βTyr(Me), displayed the monoexponential fluorescence decay. The biexponential fluorescence decay observed for Ac-βTyr(Me) is assumed to be the result of the presence of two low-energy conformations (extended and with an intramolecular hydrogen bond). Higher quenching of the fluorescence of β-tyrosine derivatives by the N-acetyl group than by the N-methylamide group moved farther was found, contrary to the data found for the respective derivatives of natural tyrosine. The obtained photophysical data are discussed with theoretical calculations (AMBER, AM1) on the basis of the rotamer model.

Thin-Layer chromatographic separation of the Z and E rotational isomers of α-N-nitroso-N-alkylamino acids

Abstract Thin-layer chromatography at 0–2 C affords a simple and rapid method for the detection and separation of the Z and E isomers of N-nitroso-N-alkylamino acids. The procedure enables conformational purity of the crystals to be determined and conformational Z⇌E interconversions to be studied.