Role of electrostatics in the binding of charged metallophthalocyanines to neutral and charged phospholipid membranes

Binding of the cationic tetra(tributylammoniomethyl)-substituted hydroxoaluminum phthalocyanine (AlPcN₄) to bilayer lipid membranes was studied by fluorescence correlation spectroscopy (FCS) and intramembrane field compensation (IFC) methods. With neutral phosphatidylcholine membranes, AlPcN₄ appeared to bind more effectively than the negatively charged tetrasulfonated aluminum phthalocyanine (AlPcS₄), which was attributed to the enhancement of the coordination interaction of aluminum with the phosphate moiety of phosphatidylcholine by the electric field created by positively charged groups of AlPcN₄. The inhibitory effect of fluoride ions on the membrane binding of both AlPcN₄ and AlPcS₄ supported the essential role of aluminum-phosphate coordination in the interaction of these phthalocyanines with phospholipids. The presence of negative or positive charges on the surface of lipid membranes modulated the binding of AlPcN₄ and AlPcS₄ in accord with the character (attraction or repulsion) of the electrostatic interaction, thus showing the significant contribution of the latter to the phthalocyanine adsorption on lipid bilayers. The data on the photodynamic activity of AlPcN₄ and AlPcS₄ as measured by sensitized photoinactivation of gramicidin channels in bilayer lipid membranes correlated well with the binding data obtained by FCS and IFC techniques. The reduced photodynamic activity of AlPcN₄ with neutral membranes violating this correlation was attributed to the concentration quenching of singlet excited states as proved by the data on the AlPcN₄ fluorescence quenching.     

Structural anatomy of telomere OB proteins

Telomere DNA-binding proteins protect the ends of chromosomes in eukaryotes. A subset of these proteins are constructed with one or more OB folds and bind with G+T-rich single-stranded DNA found at the extreme termini. The resulting DNA-OB protein complex interacts with other telomere components to coordinate critical telomere functions of DNA protection and DNA synthesis. While the first crystal and NMR structures readily explained protection of telomere ends, the picture of how single-stranded DNA becomes available to serve as primer and template for synthesis of new telomere DNA is only recently coming into focus. New structures of telomere OB fold proteins alongside insights from genetic and biochemical experiments have made significant contributions towards understanding how protein-binding OB proteins collaborate with DNA-binding OB proteins to recruit telomerase and DNA polymerase for telomere homeostasis. This review surveys telomere OB protein structures alongside highly comparable structures derived from replication protein A (RPA) components, with the goal of providing a molecular context for understanding telomere OB protein evolution and mechanism of action in protection and synthesis of telomere DNA.     

Local Conformation and Dynamics of Isoleucine in the Collagenase Cleavage Site Provide a Recognition Signal for Matrix Metalloproteinases

The mechanism by which enzymes recognize the “uniform” collagen triple helix is not well understood. Matrix metalloproteinases (MMPs) cleave collagen after the Gly residue of the triplet sequence Gly∼[Ile/Leu]-[Ala/Leu] at a single, unique, position along the peptide chain. Sequence analysis of types I-III collagen has revealed a 5-triplet sequence pattern in which the natural cleavage triplets are always flanked by a specific distribution of imino acids. NMR and MMP kinetic studies of a series of homotrimer peptides that model type III collagen have been performed to correlate conformation and dynamics at, and near, the cleavage site to collagenolytic activity. A peptide that models the natural cleavage site is significantly more active than a peptide that models a potential but non-cleavable site just 2-triplets away and NMR studies show clearly that the Ile in the leading chain of the cleavage peptide is more exposed to solvent and less locally stable than the Ile in the middle and lagging chains. We propose that the unique local instability of Ile at the cleavage site in part arises from the placement of the conserved Pro at the P₃ subsite. NMR studies of peptides with Pro substitutions indicate that the local dynamics of the three chains are directly modulated by their proximity to Pro. Correlation of peptide activity to NMR data shows that a single locally unstable chain at the cleavage site, rather than two or three labile chains, is more favorable for cleavage by MMP-1 and may be the determining factor for collagen recognition.     

On the rotational brownian motion of a bacterial idle motor. II. Theory of fluorescence correlation spectroscopy

The photon flux autocorrelation function of a fluorescent label attached to a bacterial motor shaft is calculated for the case in which the bacterial motor is considered to be actively but idly rotating. It is shown that even when the fluorescent label has a very short lifetime, fluorescence correlation spectroscopy should provide a useful tool for determining the rate of revolution of the bacterial motor under various solution conditions.     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

On the Application of Fuzzy Decision Theory in Chemotherapy

1IntreductionTheliteratUreonmulti-Criteriondecisionmaking(MCDM)problemshas~tremendouslyintherecentpast.TwomajorareashaveevolvedwhiChbothconcentrateondecisionmakingwithseveralcriteria:multiobjectivedecisionmaking(MODM)andmulti-attributedecisionmaking(MADM).TheformerconcentratesoncontinuousdecisionspaceandthelatterfocusesonproblemswithdiscreteSPace.FuzzysettheoryhascontributedtoMODMproblemsaswellastheMADMProblems.ThegeneralMODMproblemcanbedeft.edLllasfollows:Twostagescangenerallybe…     

X-ray absorption spectroscopy of selenium-containing amino acids

Received: 2 April 1999 / Accepted: 17 September 1999