Small angle neutron scattering from lysozyme solutions in unsaturated and supersaturated states (SANS from lysozyme solutions)

Small angle neutron scattering (SANS) method was used to study lysozyme solutions, with particular interest in an understanding of the crystallization process at the initial stage. It is found that (1) in the unsaturated solution, the protein molecules aggregate with a continuous increase in size when NaCl concentration is increased, and (2) in the supersaturated solution, an irreversible change, superimposed on the former process, occurs when the supersaturation is realized. These facts indicate the usefulness of SANS in detecting changes of protein molecules in solution on the nanometer scale. The reliability of the SANS results are indicated by (1) comparing them with those of small angle X-ray scattering (SAXS), and (2) comparing the effect of D(2)O and H(2)O as solvent. Since the interparticle interaction is essential in the crystallization process and a simple Guinier plot analysis is not allowed, a more rigorous framework of analyzing data with interference function is developed, through which both average interparticle distance and particle size are estimated.     

Controling the rate of protein release from polyelectrolyte complexes

Extended protein release from readily prepared, water-insoluble complexes with oppositely charged polyions is explored. Using hen egg-white lysozyme as a model, its sustained release from such complexes with a number of polyanions under physiological conditions has been demonstrated and rationalized. The rate of release varies orders of magnitude and is controlled by the nature of the polyanion (decreasing upon increase in its linear charge density, length, and hydrophobicity) and the complex particle size (the larger the particles, the slower the release).     

SANS (USH1G) regulates pre-mRNA splicing by mediating the intra-nuclear transfer of tri-snRNP complexes

Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome—the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.     

Hydration of lysozyme as observed by infrared spectrometry

Infrared spectra of a film of lysozyme 3 mum thick, immersed in an atmosphere displaying a relative humidity, or hygrometry, which spans the whole range from 0 to 1 at room temperature, are recorded. The evolution of the spectra with this relative humidity is quantitatively analyzed on the basis of a newly proposed method. It allows the precise measurement of the quantity of water that remains embedded inside the dried sample at each stage of hydration, and the definition, in terms of chemical reactions of the three hydration mechanisms that correspond to the three hydration spectra on which all experimental spectra can be decomposed. With respect to preceding similar studies, some refinements are introduced that allow improvement of the interpretation, but that also raise some new questions, which mainly concern the structure of the hydrogen-bond network around the carbonyl peptide groups.     

Engineering of human lysozyme as a polyelectrolyte by the alteration of molecular surface charge

The surface positive charges of human lysozyme were either increased or decreased to alter the electrostatic interaction between enzyme and substrate in the lytic action of human lysozyme using site-directed mutagenesis. The amino acid substitutions accompanying either the addition or the removal of two units of positive charge have shifted the optimal ionic strength (NaCl concentration in 10 mM Mes buffer, pH 6.2) for the lysis of Micrococcus lysodeikticus cell from 0.04 M to 0.1 M and from 0.04 M to 0.02 M respectively. In addition to the change in ionic strength-activity profile, the pH-activity profile and the effect of a polycationic electrolyte, poly-L-Lys-HCl, on the lytic activity were significantly changed. Owing to the shifts in both ionic strength profiles and pH profiles the Arg74/Arg126 mutant has become a better catalyst than wild-type enzyme under the conditions of high ionic strength and high pH, and the Gln41/Ser101 mutant has become a better catalyst under the conditions of low ionic strength and low pH.     

Affinity precipitation of monoclonal antibodies by nonstoichiometric polyelectrolyte complexes

The nonstoichiometric polyelectrolyte complex (PEC) formed by poly(methacrylic acid) (degree of polymerization 1830) (PMAA)and poly(N-ethyl-4-vinyl-pyridinium bromide) (degree of polymerization 530) (PEVP) undergoes reversible precipitation from aqueous solution at any desired pH-value in the range 4.5–6.5 depending on the ionic strength and PEVP/PMAA ratio in the complex. The antigen, inactivated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit was covalently coupled to PEVP. The resulting GAPDH–PEVP/PMAA complex was used for the purification of antibodies from a 6G7 clone specific towards inactivated GAPDH. The crude extract was incubated with GAPDH-containing PEC and the precipitation of the PEC was carried out at 0.01 M NaCl and pH 4.5, 5.3, 6.0 and 6.5 using PEC with PEVP/PMAA ratios of 0.45, 0.3, 0.2 and 0.15, respectively. Purified antibodies were eluted at pH 4.0 where PECs of all compositions used were insoluble.PEC precipitation is accompanied only by small nonspecific coprecipitation of proteins. Precipitated PEC could be dissolved at pH 7.3 and used repeatedly. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immobilization of invertase by encapsulation in polyelectrolyte complexes.

Free and polystyrene-bound invertase from Saccharomyces cerevisiae were encapsulated within symplex membranes which were composed of cellulose sulfate as the polymeric anion and poly(dimethyldiallylammonium chloride) as the polymeric cation. The kinetics and the performance of the encapsulated enzyme preparations have been compared to the free enzyme employing the hydrolysis of sucrose. The pH and temperature optima were only slightly affected by the encapsulation. The kinetic constants, however, were changed by the encapsulation as a result of diffusional limitation. Encapsulated invertase showed a high storage stability and a high operational stability if low substrate concentrations were applied. The coimmobilization of invertase with living cells, which are not capable of utilizing sucrose, in the described capsules, opens many possibilities in fermentation technology.     

Fluorescamine-labelled cortical fragments as a substrate for lysozyme and initiation protein (spore-lytic enzyme)

A fluorescamine assay for the detection of a spore-lytic enzyme from Clostridium perfringens is described. The substrate is prepared by treatment of cortical fragments with fluorescamine which reacts with amino terminal groups in the peptidoglycan which are not cross-linked, presumably diaminopimelic acid. Treatment of the labelled substrate with lytic enzymes results in the release of soluble fluorescent products which can be easily measured in a basic fluorometer. The assay is very sensitive, inexpensive and reproducible. As little as 1 μg of lysozyme can be detected by this assay.     

Extended, relaxed, and condensed conformations of hyaluronan observed by atomic force microscopy

The conformation of the polysaccharide hyaluronan (HA) has been investigated by tapping mode atomic force microscopy in air. HA deposited on a prehydrated mica surface favored an extended conformation, attributed to molecular combing and inhibition of subsequent chain recoil by adhesion to the structured water layer covering the surface. HA deposited on freshly cleaved mica served as a defect in a partially structured water layer, and favored relaxed, weakly helical, coiled conformations. Intramolecularly condensed forms of HA were also observed, ranging from pearl necklace forms to thick rods. The condensation is attributed to weak adhesion to the mica surface, counterion-mediated attractive electrostatic interactions between polyelectrolytes, and hydration effects. Intermolecular association of both extended and condensed forms of HA was observed to result in the formation of networks and twisted fibers, in which the chain direction is not necessarily parallel to the fiber direction. Whereas the relaxed coil and partially condensed conformations of HA are relevant to the native structure of liquid connective tissues, fully condensed rods may be more relevant for HA tethered to a cell surface or intracellular HA, and fibrous forms may be relevant for HA subjected to shear flow in tight intercellular spaces or in protein-HA complexes.     

Recovery of a charged-fusion protein from cell extracts by polyelectrolyte precipitation

Beta-galactosidase served as a model system to explore the feasibility of enhancing the selectivity of a low-cost, easily scaled separation method-precipitation. Enhanced selectivity was sought by fusing the enzyme with polypeptide tails including 5 and 11 aspartates. The unfused protein could not be selectively removed from the Escherichia coli cell extract by precipitation with polyethylenimine (PEI), but the longest fusion could be selectively removed. The presence of nucleic acids limited the purification attainable. Pretreatment with nuclease followed by diafiltration resulted in an extract from which the same fusion could be precipitated with greater than fivefold enrichment, while the untailed enzyme remained unenriched by the same precipitation step. Selectivity is attributed to the binding strength of the polyanionic tails to the polycationic PEI.     

Sorption of wheat seed lectin by polyelectrolyte complexes of chitosan

The possible application of polyelectrolyte complexes (PEC) of chitosan and copolymers of maleic acid with N-vinylpyrrolidone, styrene, and ethylene and/or chitin cross-linked sorbents (CCLS) synthesized on the basis of PEC for sorption of wheat germ agglutinin (WGA) was studied. The synthesis of spherically granulated sorbents was shown. Compared to unmodified chitosan, there was a significant increase in sorption of WGA by the PEC: PEC, 2.5-fold, and CCLS, from 3.5-fold to 7-fold.     

Water-coupled low-frequency modes of myoglobin and lysozyme observed by inelastic neutron scattering.

Conformational changes of proteins often involve the relative motion of rigid structural domains. Normal mode analysis and molecular dynamics simulations of small globular proteins predict delocalized vibrations with frequencies below 20 cm(-1), which may be overdamped in solution due to solvent friction. In search of these modes, we have studied deuterium-exchanged myoglobin and lysozyme using inelastic neutron scattering in the low-frequency range at full and low hydration to modify the degree of damping. At room temperature, the hydrated samples exhibit a more pronounced quasielastic spectrum due to diffusive motions than the dehydrated samples. The analysis of the corresponding lineshapes suggests that water modifies mainly the amplitude, but not the characteristic time of fast protein motions. At low temperatures, in contrast, the dehydrated samples exhibit larger motional amplitudes than the hydrated ones. The excess scattering, culminating at 16 cm(-1), is suggested to reflect water-coupled librations of polar side chains that are depressed in the hydrated system by strong intermolecular hydrogen bonding. Both myoglobin and lysozyme exhibit ultra-low-frequency modes below 10 cm(-1) in the dry state, possibly related to the breathing modes predicted by harmonic analysis.     

The interactions of the ruthenium(II)-cymene complexes with lysozyme and cytochrome c

JBIC Journal of Biological Inorganic Chemistry - The reactions of four cymene-capped ruthenium(II) compounds with pro-apoptotic protein, cytochrome c (Cyt), and anti-proliferative protein lysozyme...     

Crystal structures of hen egg-white lysozyme complexes with gadolinium(III) and gadolinium(III)-N-acetyl-D-glucosamine.

Analysis at 0.25 nm resolution of the crystal structures of lysozyme-Gd(III) and lysozyme-Gd(III)-N-acetyl-D-glucosamine (GlcNac), prepared by diffusion methods, show that there are two main binding positions for Gd(III), one of which is close to glutamic acid-35 and the other close to aspartic acid-52. The two sites are 0.36 nm part. There is no evidence for the weak binding of Gd(III) to any of the eight other carboxy groups of lysozyme. In the presence of Gd(III), the binding of GlcNac is similar to that observed for the binding of the beta-anomer in subsite C. There are numerous small conformational changes in the protein on binding (Gd(III) and the sugar, and these have been quantified to a first approximation by real-space refinement. These changes are similar in both structures, and involve, among other small movements, shifts of one of the disulphide bridges by up to 0.05 nm. The movement of residues 70--74 observed in the binary complex of lysozyme-GlcNac [Perkins, Johnson, Machin & Phillips (1978) Biochem. J. 173-617] is not observed in the ternary complex of lysozyme-Gd(III)-GlcNac. The nature of the lysozyme-Gd(III) complex is discussed in the light of evidence from other crystallographic studies and n.m.r. solution studies. Preliminary findings for a lysozyme-Gd(III) complex prepared by co-crystallization methods are reported.     

Binding of hyaluronan to lysozyme at various pHs and salt concentrations.

Binding of hyaluronan (HA) to lysozyme immobilized on Sepharose-6B was investigated as a function of pH and NaCl concentration. High affinity binding (Kd = 1.0-2.0 x 10(-8) M) was observed at pH 7.5 and at 10-50 mM NaCl; the number of moles of HA bound to lysozyme was twice as high at 30 mM NaCl as at 10 mM. No specific binding was observed at and above 100 mM NaCl. Binding was suppressed in the presence of chaotropic agents such as guanidinium chloride and urea. These results suggest that binding between HA and lysozyme can occur in the extracellular matrix where an electrolyte concentration as low as 50 mM could be expected due to ionic exclusion by the highly negative charge concentration arising from the polyanions present.