Mutational analysis of m-values as a strategy to identify cold-resistant substructures of the protein ensemble

Characterizing the native ensemble of protein is an important yet difficult objective of structural biology. The structural dynamics of protein macromolecules play key roles in biological function, but the short lifetimes and low population of near-native states of the protein ensemble limit their ability to be studied directly. In part to address such issues, it was shown recently that the cooperative substructures that populate a protein ensemble could be ascertained by NMR methods performed at very cold temperatures. What is presented here is an argument that these same substructures can also be determined by denaturant-induced unfolding studies performed on protein at room temperature. Data supporting this argument are given for Staphylococcal nuclease, chymotrypsin inhibitor 2, and ubiquitin. The observation of an agreement between the thermodynamics of the protein ensemble simulated under very cold temperatures to the apparent sensitivity of the ensemble to chemical denaturants at room temperature also suggests that the overall structural-thermodynamic character of an ensemble is surprisingly robust and preserved even in the presence of strong denaturing conditions.     

Molecular mechanisms of pH-driven conformational transitions of proteins: insights from continuum electrostatics calculations of acid unfolding

The acid unfolding of staphylococcal nuclease (SNase) is very cooperative (Whitten and García-Moreno, Biochemistry 2000;39:14292-14304). As many as seven hydrogen ions (H+) are bound preferentially by the acid-unfolded state relative to the native (N) state in the pH range 3.2-3.9. To investigate the mechanism of acid unfolding, structure-based pKa calculations were performed with a variety of continuum electrostatic methods. The calculations reproduced successfully the H+ binding properties of the N state between pH 5 and 9, but they systematically overestimated the number of H+ bound upon acid unfolding. The calculated pKa values of all carboxylic residues in the N state were more depressed than they should be. The discrepancy between the observed and the calculated H+ uptake upon acid unfolding was not improved by using high protein dielectric constants, structures relaxed with molecular dynamics, or other empirical modifications implemented previously by others to maximize agreement between measured and calculated pKa values. This suggests an important role for conformational fluctuations of the backbone as important determinants of pKa values of carboxylic groups. Because no global or subglobal conformational changes have been observed previously for SNase under acidic conditions above the acid-unfolding region, these fluctuations must be local. The acid unfolding of SNase does not seem to involve the disruption of the N state by accruement of intramolecular repulsive interactions, nor the protonation of key ion paired carboxylic residues. It is more consistent with modest contributions from many H+ binding groups, with an important role for local conformational fluctuations in the coupling between H+ binding and the global structural transition.     

Structure, function, and motility of vacuoles in filamentous fungi

Current information on the structure and function of motile tubular vacuoles in Pisolithus tinctorius and other fungi is reviewed. The use of fluorochromes to label the vacuole lumen is evaluated and observations on the structure and motility of vacuoles in P. tinctorius are differentiated from possible artifacts. The styryl dyes FM4-64 and MDY-64, used in yeast to demonstrate endocytosis, show little or no labeling of internal membranes in undamaged P. tinctorius cells. This agrees with our data showing that other probes for endocytosis such as Lucifer yellow CH are not taken up by hyphal tip cells. Overall, the observations do not support endocytosis in hyphal tips. It has been suggested that tubular vacuole systems carry out longitudinal transport, and evidence in favor of this hypothesis is evaluated. New data are presented to show that many of the large vacuoles in subapical cells are attached to the plasma membrane and are relatively immobile, while video sequences show movement of fluorochrome in pulses along a series of several large vacuoles, all interconnected via tubules. Tubular vacuoles from thick sections of hyphae processed under anhydrous conditions are shown by X-ray microanalysis to contain relatively high levels of P and K, as seen previously in the larger vacuoles. These results provide further evidence for a role of the tubular vacuoles in longitudinal transport of P. Copyright 1998 Academic Press.     

In vitro superoxide activity in the haemolymph of the West Indian leaf cockroach,Blaberus discoidalis

The respiratory burst is an NADPH oxidase-driven reduction of molecular oxygen to superoxide, which can occur in phagocytic cells as part of an antimicrobial defence, and is well documented among the vertebrates. This paper describes a process resembling the respiratory burst, which occurs in the haemolymph and haemocytes of the cockroach, Blaberus discoidalis. The in vitro reduction of nitroblue tetrazolium by superoxide to formazan was measured spectrophotometrically in B. discoidalis haemolymph in response to various immune elicitors. Nitroblue tetrazolium reduction was partly impeded in the presence of superoxide dismutase, a specific antioxidant which converts superoxide to hydrogen peroxide, as well as by chemicals known to inhibit the respiratory burst in vertebrates (trifluoperazine, diphenylene iodonium, and N-ethylmaleimide). This suggests the generation of superoxide anions by haemolymph as part of an immune response. Furthermore, formazan staining of elicitor-treated haemocytes was observed microscopically, with less intense staining in the presence of superoxide dismutase. Finally, respiratory burst inhibitors and superoxide dismutase enhanced the growth of E. coli incubated in whole haemolymph, implying a role for haemolymph-derived superoxide in antibacterial defence.     

Tissue distribution of migration inhibitory factor and inducible nitric oxide synthase in falciparum malaria and sepsis in African children

Background

The inflammatory nature of falciparum malaria has been acknowledged since increased circulating levels of tumour necrosis factor (TNF) were first measured, but precisely where the mediators downstream from this prototype inflammatory mediator are generated has not been investigated. Here we report on the cellular distribution, by immunohistochemistry, of migration inhibitory factor (MIF) and inducible nitric oxide synthase (iNOS) in this disease, and in sepsis.

Methods

We stained for MIF and iNOS in tissues collected during 44 paediatric autopsies in Blantyre, Malawi. These comprised 42 acutely ill comatose patients, 32 of whom were diagnosed clinically as cerebral malaria and the other 10 as non-malarial diseases. Another 2 were non-malarial, non-comatose deaths. Other control tissues were from Australian adults.

Results

Of the 32 clinically diagnosed cerebral malaria cases, 11 had negligible histological change in the brain, and no or scanty intravascular sequestration of parasitised erythrocytes, another 7 had no histological changes in the brain, but sequestered parasitised erythrocytes were present (usually dense), and the remaining 14 brains showed micro-haemorrhages and intravascular mononuclear cell accumulations, plus sequestered parasitised erythrocytes. The vascular walls of the latter group stained most strongly for iNOS. Vascular wall iNOS staining was usually of low intensity in the second group (7 brains) and was virtually absent from the cerebral vascular walls of 8 of the 10 comatose patients without malaria, and also from control brains. The chest wall was chosen as a typical non-cerebral site encompassing a range of tissues of interest. Here pronounced iNOS staining in vascular wall and skeletal muscle was present in some 50% of the children in all groups, including septic meningitis, irrespective of the degree of staining in cerebral vascular walls. Parasites or malarial pigment were rare to absent in all chest wall sections. While MIF was common in chest wall vessels, usually in association with iNOS, it was absent in brain vessels.

Conclusions

These results agree with the view that clinically diagnosed cerebral malaria in African children is a collection of overlapping syndromes acting through different organ systems, with several mechanisms, not necessarily associated with cerebral vascular inflammation and damage, combining to cause death.