Ramachandran analysis of conserved glycyl residues in homologous proteins of known structure

High conservation of glycyl residues in homologous proteins is fairly frequent. It is commonly understood that glycine tends to be highly conserved either because of its unique Ramachandran angles or to avoid steric clash that would arise with a larger side chain. Using a database of aligned 3D structures of homologous proteins we identified conserved Gly in 288 alignment positions from 85 families. Ninety-six of these alignment positions correspond to conserved Gly residue with (phi, ) values allowed for non-glycyl residues. Reasons for this observation were investigated by in-silico mutation of these glycyl residues to Ala. We found in 94% of the cases a short contact exists between the C atom of the introduced Ala with the atoms which are often distant in the primary structure. This suggests the lack of space even for a short side chain thereby explaining high conservation of glycyl residues even when they adopt (phi, ) values allowed for Ala. In 189 alignment positions, the conserved glycyl residues adopt (phi, ) values which are disallowed for Ala. In-silico mutation of these Gly residues to Ala almost always results in steric hindrance involving C atom of Ala as one would expect by comparing Ramachandran maps for Ala and Gly. Rare occurrence of the disallowed glycyl conformations even in ultrahigh resolution protein structures are accompanied by short contacts in the crystal structures and such disallowed conformations are not conserved in the homologues. These observations raise the doubt on the accuracy of such glycyl conformations in proteins.

An automated pipette puller for fabrication of glass micropipettes

Glass micropipettes are versatile probing tools for performing micro-and nano-manipulation tasks. This paper presents the design and development of an automated pipette puller system for fabrication of glass micropipettes. The pipette puller employs a new strategy for fabrication of micropipettes that enables achieving independent control of their taper, tip diameter, and bend-angle, and also facilitates theoretical derivation of simple, approximate relationships between the pipette shape and the pulling parameters. Subsequently, the design and fabrication of the pipette puller is described, which include that of the pipette heating system, the mechanical motion stages, and the control electronics of the pipette puller. The fabricated pipette puller is experimentally evaluated to demonstrate control of the taper, tip diameter, and the bend-angle of the micropipette. Further, the dependence of the taper and tip diameter on the pulling parameters is evaluated and is shown to be in alignment with the proposed theoretical relationships. (C) 2014 AIP Publishing LLC.