Rigidity of human alpha-fetoprotein tertiary structure is under ligand control

Comparative study of the natural ligand effect on structural properties and conformational stability of human alpha-fetoprotein (AFP) and its homologue, human serum albumin (HSA), was performed using several approaches, including circular dichroism, fluorescence spectroscopy, and scanning microcalorimetry. Here we show that denaturation of AFP, induced by the increase of temperature or urea concentration, is irreversible. We have established the fact that this irreversibility is caused by ligand release from the AFP molecule. Interestingly, the ligand-free form of AFP has no rigid tertiary structure but exhibits substantial secondary structure and high compactness. This means that the rigid tertiary structure of AFP is controlled by interaction with ligands, while their release results in transition of a protein molecule into a molten globule-like intermediate. In contrast, processes of HSA denaturation and unfolding are completely reversible. Release of ligands from HSA results only in a small decrease in stability but not transformation into the molten globule state.     

Triple point mutation Asp10 -> His, Asn101 -> Asp, Argl48 -> Ser in T4 phage lysozyme leads to the molten globule

The triple amino acid replacement (Asp10 His, Asn101 Asp,Arg148 Ser) in T4 phage lysozyme was carried out by site-directedmutagenesis. At acid pH (2.7) the mutant is in a confonnationalstate with the properties of the molten globule: (i) the mutantprotein molecule is essentially compact; (ii) its CD spectrumin the near UV region is drastically reduced in intensity ascompared with the wild type protein spectrum; (iii) the CD spectrumin the far UV region indicates the presence of pronounced secondarystructure in the mutant; (iv) unlike the wild type protein themutant protein can bind the hydrophobic fluorescent probe, ANS.     

Effect of natural ligands on the structural properties and conformational stability of proteins

The effect of natural ligands on the structural properties and conformational stability of proteins is reviewed. It is shown that the range of possible structural transformations induced in a protein molecule by ligand release is very wide and virtually does not depend on the nature of the protein or that of the ligands. Ligand-free forms of protein are classified from the viewpoint of structural property changes of a protein molecule.     

Circularly permuted dihydrofolate reductase of E.coli has functional activity and a destabilized tertiary structure

Three circularly permuted variants of Escherichia coli dihydrofolatereductase genes were constructed. Linkers coding tri- and pentapeptideswere used to connect the natural 5'- and 3'-terminal ends. Onlyone variant of circularly permuted protein with tripeptide linkerand the cleavage of the peptide bond between 107 and 108 aminoacid residues was produced in a good yield. The expressed proteinwas insoluble in the cells, but at pH 8.0 and higher the isolatedprotein was soluble. Enzymatic assay and physical studies haveshown that permuted dihydrofolate reductase has a destabilizedtertiary structure. Only the addition of the natural substratesor Inhibitors lead to the protein with the native-like structureand functional activity.     

Retro-MoRFs: Identifying Protein Binding Sites by Normal and Reverse Alignment and Intrinsic Disorder Prediction

Many cell functions in all living organisms rely on protein-based molecular recognition involving disorder-to-order transitions upon binding by molecular recognition features (MoRFs). A well accepted computational tool for identifying likely protein-protein interactions is sequence alignment. In this paper, we propose the combination of sequence alignment and disorder prediction as a tool to improve the confidence of identifying MoRF-based protein-protein interactions. The method of reverse sequence alignment is also rationalized here as a novel approach for finding additional interaction regions, leading to the concept of a retro-MoRF, which has the reversed sequence of an identified MoRF. The set of retro-MoRF binding partners likely overlap the partner-sets of the originally identified MoRFs. The high abundance of MoRF-containing intrinsically disordered proteins in nature suggests the possibility that the number of retro-MoRFs could likewise be very high. This hypothesis provides new grounds for exploring the mysteries of protein-protein interaction networks at the genome level.