Use of micelles in studying drug-binding sites: simulation of the tubulin-bound fluorescence of colchicine

Enhancement of the fluorescence intensity of colchicine occurs in media of low polarity and appreciable viscosity; this is suggested to be the basis of the intensification of its fluorescence when it is bound to and immobilized in tubulin. We show here that the tubulin-bound fluorescence features of colchicine are largely reconstructed upon solubilizing it in chosen micellar aggregates that offer optimal polarities and microviscosities. Triton X-100 and bile salt micelles intensify the colchicine emission but the maximal effects are obtained with tetrameric aggregates of the peptide melittin. Estimates of the polarity, microviscosity and binding-site dimensions of colchicine are obtained using this mimetic approach. Our results suggest that well chosen micellar systems act as good models to reconstruct and analyze the spectral properties of molecules immobilized in their binding sites.     

Origin of life: A hypothesis for the origin of adaptor-mediated ordered synthesis of proteins and an explanation for the choice of terminating codons in the genetic code

Life can be defined as a system of self-sustained chemical processes springing from the ordered synthesis of proteins directed by nucleic acids. To the notoriously difficult problem of the origin of this basic process of nucleic acid-directed protein synthesis, we give a solution of molecular interactions between pentanucleotides and amino acids. A particular conformation of a pentanucleotide forms a double sided template, with its ‘inside’ capable of nestling an amino acid while the ‘outside’ acts as an adaptor to a ‘codon’ triplet on long-chain nucleic acids. This serves as a primitive decoding system. An important aspect of our postulate is that a dynamic interaction is triggered, by this decoding system, through which amino acids are brought to juxtaposition facilitating peptide bond formation. Almost all the important and unique features of contemporary protein-synthesizing machinery are seen to be a direct and natural consequence of our postulate. The emergence of the termination codons also fits in, as a natural consequence of this molecular mechanism.     

Calcium ion binding to delta- and to beta-crystallins. The presence of the "EF-hand" motif in delta-crystallin that aids in calcium ion binding

Abnormal levels of endogenous calcium ions are known to induce eye lens opacity, and a variety of causative factors has been proposed, including calcium-mediated aggregation and precipitation of the lens proteins crystallins. We have specifically looked in some detail at the interaction of Ca2+ with various crystallins and its consequences. Lenses incubated in solutions containing 10 mM Ca2+ or 5 mM Tb3+ opacified. Fluorescence titration of crystallins with TbCl3 revealed that this ion binds to delta- and beta-crystallins in solution. Equilibrium dialysis showed that four Ca2+ ions bind to one delta-crystallin tetramer with an affinity of 4.3 x 10(3) M-1. Analysis of the amino acid sequence of delta-crystallin reveals the presence of a calmodulin-type "helix-loop-helix" or "EF-hand" calcium ion binding conformational motif in the region comprising residues 300-350. This is a novel feature of the molecule not reported so far. No other crystallins appear to have this motif. beta-Crystallin also binds four Ca2+ ions/aggregate unit of mass 160 kDa, with an affinity of 2.6 x 10(3) M-1, presumably in the midregion of the molecule that is rich in anionic and polar residues. Circular dichroism spectroscopy shows that the binding of calcium ion leads to subtle conformational changes in the molecules, notably in the tertiary structure.     

Two forms of acid alpha-D-mannosidase in monkey brain: evidence for the co-existence of high mannose and complex oligosaccharides in one form

Lysosomal alpha-D-mannosidase of monkey brain existed in two forms. One form of mannosidase was bound to the Ricinus communis agglutinin120 (RCA1)-Sepharose and could be specifically eluted with lactose. The other form did not bind to the RCA1-Sepharose. Both forms of mannosidase could bind to a similar extent to the immobilized brain lysosomal receptor protein. Both the forms were purified to apparent homogeneity. Neutral sugar analysis by GLC showed the presence of glucose, mannose and galactose in the RCA1-Sepharose bindable mannosidase and glucose and mannose in the non-bindable mannosidase. Several other brain lysosomal hydrolases did not bind to the RCA1-Sepharose. The results suggested the existence of only high mannose oligosaccharides in the RCA1 non-bindable mannosidase and both high mannose and complex oligosaccharides in the bindable mannosidase.     

Effect of protein denaturants on the structure of water: Electron spin resonance spin probe study

The effect of the urea-class of protein denaturants on the structure of liquid water was studied. The method chosen was to monitor the microviscosity of the medium by estimating the reorientational correlation time, τ₈ and proton hyperfine linewidth, WH of an inert stable organic free radical (2, 2, 6, 4-tetramethyl-piperid 4-one-l-oxyl) by measuring its electron spin resonance spectra in aqueous denaturant solutions. Urea, thiourea and guanidinium chloride were found to disrupt water structure efficiently and continuously, with the effect significant at low molarities. Dimethyl urea was found to be somewhat less efficient. The relation between the structure-breaking tendency of the denaturants and their ability to weaken hydrophobic interactions is rationalised.     

A new type of representation of dipeptide conformation.

The possibility of representing the conformations of a pair of peptide units in terms of two parameters, namely angle (nu) between the two peptide planes and the virtual-bond angle delta (C(alpha)i-1, C(alpha)i, C(alpha)i+1), and its usefulness in recognizing unfavourable conformations, is discussed. To exemplify the concept, the local conformations in lysozyme have been plotted in the (nu,delta) plane.