Recombinant human hemoglobin: Expression and refolding of β-globin fromEscherichia coli

A plasmid analogous to the one described by Nagai and Thogersen (Nature,309, 810–812, 1984) has been constructed for the expression of globins inE. coli. Induction with nalidixic acid produces high yields of a fusion protein, NS1-FX--globin, where NS1 represents 81 residues of a flu virus protein and FX represents a blood-clotting Factor Xa recognition sequence, Ile-Glu-Gly-Arg. This fusion protein is readily solubilized in 50 mM NaOH and remains in solution when thepH is adjusted to 8.6. Under these conditions, the fusion protein is hydrolyzed by activated Factor X, giving authentic -globin which can be folded in the presence of cyanohemin and native -chains to produce a tetrameric hemoglobin with the functional properties of natural human hemoglobin.     

Protein aggregated into bacterial inclusion bodies does not result in protection from proteolytic digestion

Proteolytic resistance, as conferred by protein aggregation into inclusion bodies, has not been explored in detail. We have investigated the eventual digestion of several closely-related proteins, namely six insertional and two fusion mutants of the homotrimeric bacteriophage P22 tailspike (TSP) protein. When over-produced in E. coli, all these polypeptides form inclusion bodies accompanied by only traces of soluble protein. The mutations introduced in TSP impaired its degradation and enhanced its half live up to ten-fold, without affecting protein solubility. This indicates that protein properties other than solubility, are the main determinants of susceptibility to proteolysis. In addition, the analysis of the degradation fragments strongly suggests that the aggregated TSP polypeptides undergo a site-limited proteolytic attack, and that their complete digestion occurs through an in situ cascade cleavage process.     

DBF (Disulfide Bond Forming) Enzyme from the Hyperthermophilic Archaebacterium Sulfolobus Solfataricus Behaves Like a Molecular Chaperone

DBF enzyme from the hyperthermophilic archaebacterium Sulfolobus solfataricus greatly enhances the refolding at 30°C of denatured and reduced bovine pancreatic ribonuclease (Guagliardi et al., 1992). Here we show that DBF behaves like a molecular chaperone: it affects in an ATP-dependent manner the in vitro refolding at 50°C of two thermostable dehydrogenases, an alcohol dehydrogenase and a glutamate dehydrogenase from S. solfataricus. This paper also reports the complete amino acid sequence of DBF. The role of molecular chaperones from thermophilic microorganisms in applied biocatalysis is discussed.     

DBF (Disulfide Bond Forming) Enzyme from the Hyperthermophilic Archaebacterium Sulfolobus Solfataricus Behaves Like a Molecular Chaperone

DBF enzyme from the hyperthermophilic archaebacterium Sulfolobus solfataricus greatly enhances the refolding at 30°C of denatured and reduced bovine pancreatic ribonuclease (Guagliardi et al., 1992). Here we show that DBF behaves like a molecular chaperone: it affects in an ATP-dependent manner the in vitro refolding at 50°C of two thermostable dehydrogenases, an alcohol dehydrogenase and a glutamate dehydrogenase from S. solfataricus. This paper also reports the complete amino acid sequence of DBF. The role of molecular chaperones from thermophilic microorganisms in applied biocatalysis is discussed.     

Thermostability gradient in the collagen triple helix reveals its multi-domain structure

A triple-helical conformation and stability at physiological temperature are critical for the mechanical and biological functions of the fibril-forming collagens. Here, we characterized the role of consecutive domains of collagen II in stabilizing the triple helix. Analysis of melting temperatures of genetically engineered collagen-like proteins consisting of tandem repeats of the D1, D2, D3 or D4 collagen II periods revealed the presence of a gradient of thermostability along the collagen molecule with thermolabile N-terminal domains and thermostable C-terminal domains. These results imply a multi-domain character of the collagen triple helix. Assays of thermostabilities of the Arg75Cys and Arg789Cys collagen II mutants suggest that, in contrast to the thermostable domains, the thermolabile domains are able to accommodate amino acid substitutions without altering the thermostability of the entire collagen molecule.     

The effect of salts on the activity and stability of Escherichia coli and Haloferax volcanii dihydrofolate reductases

The extremely halophilic Archae require near-saturating concentrations of salt in the external environment and in their cytoplasm, potassium being the predominant intracellular cation. The proteins of these organisms have evolved to function in concentrations of salt that inactivate or precipitate homologous proteins from non-halophilic species. It has been proposed that haloadaptation is primarily due to clustering of acidic residues on the surface of the protein, and that these clusters bind networks of hydrated ions. The dihydrofolate reductases from Escherichia coli (ecDHFR) and two DHFR isozymes from Haloferax volcanii (hvDHFR1 and hvDHFR2) have been used as a model system to compare the effect of salts on a mesophilic and halophilic enzyme. The KCl-dependence of the activity and substrate affinity was investigated. ecDHFR is largely inactivated above 1M KCl, with no major effect on substrate affinity. hvDHFR1 and hvDHFR2 unfold at KCl concentrations below approximately 0.5M. Above approximately 1M, the KCl dependence of the hvDHFR activities can be attributed to the effect of salt on substrate affinity. The abilities of NaCl, KCl, and CsCl to enhance the stability to urea denaturation were determined, and similar efficacies of stabilization were observed for all three DHFR variants. The DeltaG degrees (H(2)O) values increased linearly with increasing KCl and CsCl concentrations. The increase of DeltaG degrees (H(2)O) as a function of the smallest cation, NaCl, is slightly curved, suggesting a minor stabilization from cation binding or screening of electrostatic repulsion. At their respective physiological ionic strengths, the DHFR variants exhibit similar stabilities. Salts stabilize ecDHFR and the hvDHFRs by a common mechanism, not a halophile-specific mechanism, such as the binding of hydrated salt networks. The primary mode of salt stabilization of the mesophilic and halophilic DHFRs appears to be through preferential hydration and the Hofmeister effect of salt on the activity and entropy of the aqueous solvent. In support of this conclusion, all three DHFRs are similarly stabilized by the non-ionic cosolute, sucrose.     

Heat-stable enzymes from extremely thermophilic and hyperthermophilic microorganisms

Only in the last decade have microorganisms been discovered which grow near or above 100°C. The enzymes that are formed by these extremely thermophilic (growth temperature 65 to 85°C) and hyperthermophilic (growth temperature 85 to 110°C) microorganisms are of great interest. This review covers the extracellular and intracellular enzymes of these exotic microorganisms that have recently been described. Polymer-hydrolysing enzymes, such as amylolytic, cellulolytic, hemicellulolytic and proteolytic enzymes, will be discussed. In addition, the properties of the intracellular enzymes involved in carbohydrate and amino-acid metabolism and DNA-binding and chaperones and chaperone-like proteins from hyperthermophiles are described. Due to the unusual properties of these heat-stable enzymes, they are expected to fill the gap between biological and chemical processes.The authors are with the Technical University Hamburg-Harburg, Institute of Biotechnology, Department of Technical Microbiology, Denickestrasse 15, D-21071 Hamburg, Germany     

LINUS: A hierarchic procedure to predict the fold of a protein

We describe LINUS, a hierarchic procedure to predict the fold of a protein from its amino acid sequence alone. The algorithm, which has been implemented in a computer program, was applied to large, overlapping fragments from a diverse test set of 7 X-ray-elucidated proteins, with encouraging results. For all proteins but one, the overall fragment topology is well predicted, including both secondary and supersecondary structure. The algorithm was also applied to a molecule of unknown conformation, groES, inwhich X-ray structure determination is presently ongoing. LINUS is an acronym for Local Independently Nucleated Units of Structure. The procedure ascends the folding hierarchy in discrete stages, with concomitant accretion of structure at each step. The chain is represented by simplified geometry and folds under the influence of a primitive energy function. The only accurately described energetic quantity in this work is hard sphere repulsion–the principal forceinvolved in organizing protein conformation [Richards, F. M. Ann. Rev. Biophys. Bioeng. 6:151–176, 1977]. Among other applications, the method is a natural tool for use in the human genome initiative. © 1995 Wiley-Liss, Inc.     

Protein folding

The importance of protein folding in the biosynthesis of proteins is reviewed.