15N-labeled hen lysozyme has been studied by 2D and 3D NMR in order to characterize its dynamic behavior. The resonances of all main-chain amide nitrogen atoms were assigned, as were resonances of nitrogen atoms in 28 side chains. Relaxation measurements for the main-chain and arginine and tryptophan side-chain 15N nuclei used standard methods, and those for the 15N nuclei of asparagine and glutamine side chains used pulse sequences designed to remove unwanted relaxation pathways in the NH2 groups. The calculated order parameters (S2) show that the majority of main-chain amides undergo only small amplitude librational motions on a fast time scale (S2 > or = 0.8). Increased main-chain motion (0.5 < S2 < 0.8) is observed for a total of 19 residues located at the C-terminus, in loop and turn regions, and in the first strand of the main beta-sheet. Order parameters derived for the side chains range from 0.05 to 0.9; five of the six tryptophan residues have high order parameters (S2 > or = 0.8), consistent with their location in the closely packed core of the protein, whereas the order parameters between 0.05 and 0.3 for arginine residues confirm increased side-chain mobility at the protein surface. Order parameters for the side chains of asparagine and glutamine residues range from 0.2 to 0.8; high values are found for side chains that have low solvent accessible surfaces and well-defined chi 1 values, as measured by 3J alpha beta coupling constants. Many of the main-chain and side-chain groups with low order parameters have higher than average temperature factors in X-ray crystal structures and increased positional uncertainty in NMR solution structures. They also tend to lack persistent hydrogen bond interactions and protection against amide hydrogen exchange. The most significant correlations are found between residues with low order parameters and high surface accessibility in both crystal and solution structures. The results suggest that a lack of van der Waals contacts is a major determinant of side-chain and main-chain mobility in proteins.

A novel protein-deamidating enzyme was purified to homogeneity from Chryseobacterium proteolyticum and the gene encoding it was cloned. The enzyme is a monomer with a pI of 10.0, a measured M r of < 20 000 and a calculated M r of 19 860. Extensive comparison with Streptoverticillium transglutaminase showed that the protein-deamidating enzyme lacked transglutaminase activity in terms of hydroxamateformation between benzyloxycarbonyl-Gln-Gly and hydroxylamine, or monodansylcadaverine incorporation into casein. The enzyme deamidated the two glutaminyl residues in the oxidized insulin A chain and deamidated both casein and the oxidized insulin B chain with higher catalytic efficiencies (k cat /K m ) than with short peptides. The enzyme was active against several proteins, including insoluble wheat gluten, but did not deamidate asparaginyl residues in peptides, free glutamine or other amides. The enzyme was therefore named protein-glutaminase (EC 3.5.1). The gene encoding the protein was cloned and, when expressed in Escherichia coli, the protein product had proteinglutaminase activity and cross-reacted with antiserum raised against the purified enzyme. The protein-glutaminase was shown to be expressed as a prepro-protein with a putative signal peptide of 21 amino acids and a prosequence of 114 amino acids. The amino-acid sequence had no obvious homology to any published sequence and is therefore a novel protein-glutaminase.

Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene, pdiA, encoding PDIA was previously isolated from Aspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A. pdiA also complemented PDI function in a Saccharomyces cerevisiae ⌬pdi1 mutant in a yeast-based killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA, suggesting that the induction of pdiA is not part of the primary stress response. An increased level of pdiA transcripts was also observed in two A. niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae, overexpression of PDIA did not increase secreted yields of HEWL in A. niger, suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%.

A glucoamylase ::green fluorescent protein fusion (GLA ::sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA ::sGFP which employed slightly different lengths of the glucoamylase protein (GLA499 and GLA514). Expression of GLA ::sGFP revealed that fluorescence was localized in the hyphal cell walls and septa, and that fluorescence was most intense at hyphal apices. Extracellular GLA ::sGFP was detectable by Western blotting only in the supernatant of young cultures grown in soya milk medium. In older cultures, acidification of the medium and induction of proteases were probably responsible for the loss of extracellular and cell wall fluorescence and the inability to detect GLA ::sGFP by Western analysis. A strain containing the GLA : :sGFP construct was subjected to UV mutagenesis and survivors screened for mutations in the general secretory pathway. Three mutants were isolated that were unable to form a halo on either starch or gelatin medium. All three mutants grew poorly compared to the parental strain. Fluorescence microscopy revealed that for two of the mutants, GLA ::sGFP accumulated intracellularly with no evidence of wall fluorescence, whereas for the third mutant, wall fluorescence was observed with no evidence of intracellular accumulation. These results indicate that the GLA ::sGFP fusion constructs can be used as convenient fluorescent markers to study the dynamics of protein secretion in vivo and as a tool in the isolation of mutants in the general secretory pathway.