Retrovirus integrase: identification of a potential leucine zipper motif

The secondary structure of the retrovirus integration protein(IN) was predicted from seven inferred retrovirus IN sequences.The IN sequences were aligned by computer and the phylogeneticrelationships between them were determined. The secondary structureof the aligned IN sequences was predicted by two consensus predictionmethods. The predicted secondary structural patterns from thetwo consensus prediction schemes were compared with and superimposedon a composite structural profile of hydropathic/chain flexibility/amphipathicindexes with each index profile being calculated independentlyfor the aligned IN sequences. The use of this composite structuralprofile not only enhanced the prediction accuracy but also helpedin defining the surface loop regions which would be otherwiseunpredictable by the use of consensus prediction methods alone.An amphipathic helix was identified by these united structuralprediction-chain property profiles. Helical wheel analysis gavethe amphipathic helix a coiled-coil like pattern which was similarto the leucine zipper discovered for some eukaryotic gene regulatoryproteins. The proposed amphipathic helix may play an essentialrole in defining the biological properties of IN.     

Molecular model-building of amylin and {alpha}-calcitonin gene-related polypeptide hormones using a combination of knowledge sources

Amylin is the major component of the amyloid found in the pancreasesof noninsulin-dependent diabetics (type 2 diabetes). It is a37 amino acid polypeptide and has been shown to have 46% sequenceidentity with the neuropeptide <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-calcitonin gene-related peptide(<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-CGRP). Both amylin and <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-CGRP are known to be potent inhibitorsof glycogen synthesis in stripped rat soleus muscle. Secondarystructure prediction and tertiary structure model-building showthe two polypeptides to have an <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helix/ß-strand motifsimilar to that observed in the insulin B-chain. The resultshave been supported by CD spectroscopy, although there is nosequence similarity between insulin and amylin/<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-CGRP. Aggregationstates have been predicted based on the dimeric and hexamericarrangements seen in porcine insulin. Rat and hamster amylinhave a changed sequence motif in the ß-strand whichresults in lack of amyloid formation and type 2 diabetes. This,we propose, is caused by disruption of hydrogen bonding whichprevents the formation of the dimer.     

Molecular cartography of proteins: surface relief analysis of the calf eye lens protein gamma-crystallin

Methods of calculating the protein molecular surface and differentmap representations are described. The maps are obtained byprojection of the space-filling molecular model on the surfaceof the ellipsoid of inertia. A new approach to surface analysisis proposed which is based on the use of three general maps:an identification map with all residues outlined, a surfacerelief map and a coloured map with a specific colour for eachof the surface atoms. Superposition of these maps greatly simplifiesmolecular surface analysis. The usefulness of such an approachhas been demonstrated by the study of the relief of the calfeye lens protein <IMG SRC="http://peds.oxfordjournals.org/math/gamma.gif" ALT="{gamma}" BORDER="0">-crystallin II. Protrusions of the relief havebeen shown to be occupied generally by charged residues, butin some cases by the hydrophobic ones. It is interesting tonote that in crystal medium the protruding residues are involved,in the majority of cases, in intermolecular contacts. The protrudingregions have been found to be pseudosymmetrical to each otherin accordance with the two-fold rotation axis of the molecule.However, the colours of these regions, i.e. the atoms of thecorresponding side chains, differ greatly.     

High-yield expression in Escherichia coli of soluble human alpha-hemoglobin complexed with its molecular chaperone

The alpha-subunits of human hemoglobin (Hb) have been more difficult to express than beta-chains owing to the high instability of alpha-chains. Here, we describe the production in Escherichia coli of a soluble recombinant alpha-Hb with human alpha-hemoglobin-stabilizing protein (AHSP), its molecular chaperone. To succeed in this expression, we have constructed a vector pGEX-alpha-AHSP which contains two cassettes arranged in tandem in the same orientation permitting to express alpha-hemoglobin and human AHSP. While the GST-alpha-Hb alone was expressed in E.coli as insoluble protein, even after adding lysate containing recombinant AHSP, the expression vector pGEX-alpha-AHSP permits the co-expression of soluble GST-alpha-Hb and GST-AHSP. The alpha-Hb, produced at a high yield of 12 to 20 mg per liter of culture, was then purified as a complex with its chaperone. Biochemical and biophysical properties of recombinant AHSP/recombinant alpha-Hb complex were similar to those of recombinant AHSP/native alpha-Hb complex as assessed by UV/visible and CO or O(2) binding properties. This co-expression technique can be use to study the interaction between a molecular chaperone and its target protein and, more generally, this system would be particularly interesting for the study of partner proteins when one or both proteins are individually unstable.     

Synthesis and characterization of a recombinant fragment of human {alpha}-fetoprotein with antigenic selectivity versus albumin

A DNA sequence coding for human <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-fetoprotein amino acid sequence38–119 was synthesized and cloned in a bacterial expressionvector. The <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-fetoprotein sequence was selected as the leasthomologous to albumin, since the two proteins have an overallamino acid identity of %. A chimeric protein was obtained whichwas purified by preparative electrophoresis and characterizedin its primary structure by fast atom bombardment mass spectrometry.About 70% of the <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-fetoprotein sequence was physically mappedand found to correspond to the amino acids encoded in the syntheticgene. The use of this recombinant protein allowed the selectionof monoclonal antibodies recognizing both the recombinant fragmentand native <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-fetoprotein. These antibodies should allow the developmentof an immunoassay for <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-fetoprotein with absolute selectivityversus albumin. This might result in more sensitive clinicaldeterminations, avoiding the possibility of cross-reactions.