Some aspects of structure-function relations of human hemoglobin

Contains fulltext : mmubn000001_247054704.pdf (publisher's version ) (Open Access)Promotor : G. van Os cum laude80 p

Protein Engineering and Biosynthesis of Nisin and Regulation of Transcription of the Structural nisA Gene

The lantibiotic nisin, produced by Lactococcus lactis, is an antimicrobial peptide characterized by the presence of three unsaturated amino acid side chains (two dehydroalanines and one dehydrobutyrine) and five (β-methyl)lanthionine rings, which are formed post-translationally. Nisin is widely used in the food industry as a preservative, since it inhibits the growth of unwanted gram-positive bacteria. One of the objectives of our research is to get insight in the complex biosynthesis and regulation of production of nisin. The structure and function of several biosynthetic genes were studied by making gene disruptions and by subsequently investigating their effects on nisin gene regulation, biosynthesis, secretion and immunity. An exciting finding is that nisin itself, when added to the culture medium, can induce the transcription of its own structural gene. Another goal is to design and produce altered nisin molecules with desirable properties by protein engineering. In addition to previously reported mutant nisins with improved stability, solubility or activity, recent results on the protein engineering of residues Ile1, Dhb2, AlaS3, Lys12, AbuS13, Met17, Asn20 and Met21 indicate that (i) residue 1 can be replaced without dramatic loss of activity; (ii) the presence of a Thr residue at position 2 significantly lowers the antimicrobial potency, whereas the presence of a Dha residue at position 2 improves activity; (iii) the replacement of AlaS3 by AbuS leads to a dramatic loss of activity, probably due to a conformational change in the first lanthionine ring; (iv) the integrity and hydrophobicity of ring 3 are important for antimicrobial activity; and (v) the hinge region between rings 3 and 4 is important but not essential for antimicrobial activity.

The interaction of organic phosphates with human and chicken hemoglobin

The binding properties of inositol hexaphosphate and 2,3 biphosphoglycerate to chicken and human deoxyhemoglobin and carboxyhemoglobin were compared. It appeared that in all cases the binding to chicken hemoglobin is much stronger than to human hemoglobin. This is very probably due to the fact that 4 out of the 12 residues, responsible for the binding of phosphates in chicken hemoglobin, are arginines. These are absent in human hemoglobin, where the binding site is made up by only 8 residues. For chicken hemoglobin one strong binding site could be observed in both unliganded and liganded hemoglobin. From these observations it is concluded tht the same binding site is involved in both the oxy and deoxy structure showing a different affinity to phosphates in the two conformational states. For human hemoglobin the same conclusion was reached.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering.

Nisin is a 3.4-kDa antimicrobial peptide that, as a result of posttranslational modifications, contains unsaturated amino acids and lanthionine residues. It is applied as a preservative in various food products. The solubility and stability of nisin and nisin mutants have been studied. It is demonstrated that nisin mutants can be produced with improved functional properties. The solubility of nisin A is highest at low pH values and gradually decreases by almost 2 orders of magnitude when the pH of the solution exceeds a value of 7. At low pH, nisin Z exhibits a decreased solubility relative to that of nisin A; at neutral and higher pH values, the solubilities of both variants are comparable. Two mutants of nisin Z, which contain lysyl residues at positions 27 and 31, respectively, instead of Asn-27 and His-31, were produced with the aim of reaching higher solubility at neutral pH. Both mutants were purified to homogeneity, and their structures were confirmed by one- and two-dimensional 1H nuclear magnetic resonance. Their antimicrobial activities were found to be similar to that of nisin Z, whereas their solubilities at pH 7 increased by factors of 4 and 7, respectively. The chemical stability of nisin A was studied in the pH range of 2 to 8 and at a 20, 37, and 75 degrees C. Optimal stability was observed at pH 3.0. Nisin Z showed a behavior similar to that of nisin A. A mutant containing dehydrobutyrine at position 5 instead of dehydroalanine had lower activity but was significantly more resistant to acid-catalyzed chemical degradation than wild-type nisin Z

Mechanisms of Hemoglobin Adaptation to High Altitude Hypoxia

Storz, Jay F., and Hideaki Moriyama. Mechanisms of hemoglobin adaptation. High Alt. Med. Biol. 9:148–157, 2008.—Evidence from a number of vertebrate taxa suggests that modifications of hemoglobin (Hb) function may often play a key role in mediating an adaptive response to high altitude hypoxia. The respiratory functions of Hb are a product of the protein's intrinsic O2-binding affinity and its interactions with allosteric effectors such as protons, chloride ions, CO2, and organic phosphates. Here we review several case studies involving high altitude vertebrates where it has been possible to identify specific mechanisms of Hb adaptation to hypoxia. In addition to comparative studies of Hbs from diverse animal species, functional studies of human Hb mutants also suggest that there is ample scope for evolutionary adjustments in Hb–O2 affinity through alterations of the equilibrium constants of O2 binding to deoxy- and oxyHb or through changes in the allosteric equilibrium constants for the transition between the deoxy- and oxyHb quaternary structures. It may be the case that certain evolutionary paths are followed more often than others simply because they are subject to less stringent pleiotropic constraints