Protein and Peptide Letters - Volume 9, Issue 2, 2002

In the search for increasingly accurate protein structures, technological advances are opening up new possibilities. In the last few years the wide accessibility of intense synchrotron sources, the availability of efficient 2D-detectors, and the routine use of cryo-crystallography techniques have yielded a significant number of atomic resolution protein structures. Here we review the most interesting results achieved in this field with a particular emphasis to the biological implications and to the correlation between protein geometry and conformation.

Collagen is the major structural protein in skin, bone, tendon, cartilage and blood vessels. Its triple helical structure has been long studied by fibre diffraction. More recently, single crystal X-ray diffraction on collagen-like polypeptide models has allowed a significantly improved description of the triple helix and it has shed light on the relationships between triple helix features and stability. This review outlines the current knowledge regarding collagen triple helix structure, stability, and assembly, with a particular emphasis on the latest structural results.

This review briefly introduces the principles of atomic force microscopy (AFM) applied to protein samples. AFM provides three-dimensional surface images of the proteins with high resolution. The advantage of AFM for protein studies is that AFM can visualize directly the molecule under physiological conditions without previous treatment. AFM operated in the force-spectroscopy mode is now a widespread technique, often used to investigate ligand-receptor interactions with the goal of measuring forces at the individual molecule level.

The RNA Recognition Motif (RRM) family of RNA-binding domains comprises distinct structural subclasses which can be equated to various types of cognate RNAs in relation to biological functions. By identifying structural templates within the appropriate RRM subclass, we have homology-modelled the threedimensional structure of the hermes gene-encoded RRM. Our findings lead us to propose potential RNA targets for the corresponding protein and to predict possible functions in RNA metabolism during heart development.

Zwitterionic dipeptides have recently been shown to exist in water mainly as nine conformational forms with specific combinations of backbone ψ, ω and φ torsions, which allows conformer-specific molecular recognition of peptide ligands by proteins. Here, we show that pairs of virtual backbone torsions can also define these nine conformational forms, and that comparing these virtual torsions in dipeptides with those of backbone-modified pseudopeptides offers an improved procedure for evaluating peptidomimetics for therapeutic applications.

The interaction between duodenase and inhibitors of Bowman-Birk type from soybeans (BBI) and lima beans (LBI) was investigated. Duodenase was shown to interact only with antichymotrypsin site of these inhibitors. The inhibition constants of duodenase by BBI, LBI, BBI-trypsin and LBI trypsin complexes were 4, 23, 400, 600 nM respectively.

In this study, we demonstrate that human mu-opioid receptors do form SDS-resistant homodimers and examine the ability of human mu-opioid receptors to dimerize and the role of agonists in the dimerization. Increasing concentrations and longer exposure of agonists reduce the levels of dimmer with a corresponding increase in the levels of monomer. This effect is achieved with both peptide and alkaloid opioid agonists and it is antagonist reversible. These results suggest that human muopioid receptors are present as receptor oligomers and interconversion between dimeric and monomeric forms may be important for biological activity.

In response to ligand binding and activating mutations, the lutropin receptor undergoes a conformational change to trigger a cellular response. D556 is the most common locus for naturally occurring activating mutations of the lutropin receptor, and a D556A mutant is shown to be constitutively active. A water-mediated proton transfer is postulated as part of the transmembrane signaling mechanism. Using energy minimization and ab initio calculations, a hydrogen bonding network involving a highly constrained water molecule(s) and D556 (helix 6) and N593 / N597 / Y601 (helix 7) is presented.

A lectin from the red marine alga Hypnea musciformis (HML) was purified by extraction with 20 mM PBS, precipitation with 70% saturated ammonium sulphate, ion-exchange DEAE-Cellulose chromatography and RP-HPLC. The 9.3 kDa polypeptide agglutinates erythrocytes from various sources and shows oligomerization tendencies under certain MALDI-TOF / MS conditions. Preliminary N-terminal sequencing and biological assays strongly suggest that the HML may belong to a new class of algae lectins.

An amphiphilic support was developed by the suspension polymerization of styrene and 1,4-butanediol dimethacrylate. The copolymer was converted to benzhydryl resin, BDDMA-PS-BH, by a two-step polymer analogous reaction. This resin was employed as a support for the synthesis of delta sleep inducing peptide (DSIP), Trp-Ala-Gly-Gly-Asp-Ala-Ser- Gly-Glu, in high yield and purity by Fmoc / t-Bu tactics.

The time course of Aβ fibril formation was characterized using a variety of assays. The effect of Aβ on the membrane fluidity and permeability was assessed by monitoring the anisotropy of the fluorescent probe DPH and TMA-DPH and measuring the release of vesicle-entrapped with three different molecular weight fluorescence probes (ANTS / DPX, Calcein, FD-4). The results show fibrils had formed after 4 days. Aggregated Aβmay decrease the membrane fluidity and induce the leakage of ANTS and Calcein in a dose -dependent manner. These effects may have some relation with Aβ neurotoxicity.

Atomic Force Microscopy (AFM) has been a useful tool for molecular surface analysis and to estimate topographical properties of proteins. Here we report a topographical study of a chymotrypsin inhibitor from Schizolobium parahyba seeds (SPCI) by AFM. The underlying structure of SPCI oligomers has been resolved in nanometer order resolution. SPCI oligomerize in hexagonal, ellipsoid, comet, pyramidal, and “Z“ shaped. The hexagonal was the most observed oligomer shape.