Stefania Brocca

Secondary structure, conformational stability and glycosylation of a recombinant Candida rugosa lipase studied by Fourier-transform infrared spectroscopy

The secondary structure of lipase 1 from Candida rugosa, a model system for large monomeric enzymes, has been studied by FTIR (Fourier-transform infrared) spectroscopy in water and 2H2O. The secondary structure content, determined by the analysis of the amide I band absorption through second derivative and curve fitting procedures, is in agreement with that estimated by X-ray data and predicts, in addition, the existence of two classes of alpha-helices. We have also investigated the enzyme stability and aggregation at high temperature by following the protein unfolding. The thermal stability determined by FTIR is in excellent agreement with the temperature dependence of the lipase activity. Furthermore, new insights on the glycosylation of the recombinant protein produced in Pichia pastoris and on its heterogeneity related to different fermentation batches were obtained by the analysis of the IR absorption in the 1200-900 cm(-1) carbohydrate region. A drastic reduction of the intensity of this band was found after enzymic deglycosylation of the protein. To confirm that the FTIR absorption in the 1200-900 cm(-1) region depends on the carbohydrate content and glycoform distribution, we performed an MS analysis of the protein sugar moieties. Glycosidic structures of the high mannose type were found, with mannoses ranging from 8 to 25 residues.

Cloning and analysis of Candida cylindracea lipase sequences.

Lipases (Lip) hydrolyze triglycerides into fatty acids and glycerol. Lip produced by the yeast Candida cylindracea are encoded by multiple genomic sequences. We report the molecular cloning and characterization of three genes from this family. They encode putative mature 57-kDa proteins of 534 amino acids (aa). To date, five Lip-encoding genomic sequences from C. cylindracea have been characterized in our laboratory. The five deduced aa sequences share an overall homology of 80%. These sequences have been aligned with each other and with those of homologous enzymes, the Lip from the mould Geotrichum candidum and the acetylcholinesterase from Torpedo californica, whose three-dimensional structures have been solved by X-ray analysis. The C. cylindracea Lip appear to have a structural organization similar to that described for both enzymes.

Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes

The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site. In the active enzyme conformation, the open lid participates in the substrate‐binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain‐length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium.

Physiological control on the expression and secretion of Candida rugosa lipase

The fungus Candida rugosa secretes an extracellular lipase whose production is induced by the addition of fatty acids to the culture broth. This lipase is indeed composed by several protein isoforms partly differing in their catalytic properties. Synthesis and secretion of lipase proteins by C. rugosa cells were studied in culture media containing either glucose or oleic acid as the carbon source. It was shown that, according to their regulation, lipase-encoding genes might be grouped in two classes, one of which is constitutively expressed and the other is induced by fatty acids. The synthesis of inducible enzymes is inhibited at the level of transcription by the addition of glucose and, conversely, oleic acid appears to hinder the synthesis of the constitutive lipase. Growth conditions supporting high level expression both in batch and in continuous culture give rise to the intracellular accumulation of enzyme, possibly due to the existence of a rate-limiting step in the transport of the newly synthesized protein. These results suggest the possibility to develop fermentation processes aimed at the control of the enzyme composition.

Charge-surface correlation in electrospray ionization of folded and unfolded proteins

Electrospray-ionization mass spectrometry (ESI-MS) is widely used for protein studies. It has been shown that the extent of protein ionization under nondenaturing conditions correlates well with the solvent-accessible surface area of the tridimensional structure, for either folded monomers or multimeric complexes. The goal of this study was to test whether this relation holds for unfolded proteins as well. In order to overcome the paucity of structural data, the server ProtSA was used to model the conformational ensembles of proteins in the unfolded state and generate estimates of the average solvent accessibility. The results are analyzed along with literature data or original measurements by ESI-MS. It is found that the charge-to-surface relation holds for proteins in the unfolded state, free from solvent effects. A double-log plot is derived, in close agreement with published data for folded proteins. These results suggest that the solvent-accessible surface area is a key factor determining the extent of protein ionization by electrospray, independent of the conformational state. This conclusion helps rationalizing conformational effects in protein ESI-MS. The here reported relation can be used to predict the average solvent accessibility and, hence, the state of folding of unknown proteins from ESI-MS data.

Order propensity of an intrinsically disordered protein, the cyclin-dependent-kinase inhibitor Sic1.

Intrinsically disordered proteins (IDPs) carry out important biological functions and offer an instructive model system for folding and binding studies. However, their structural characterization in the absence of interactors is hindered by their highly dynamic conformation. The cyclin‐dependent‐kinase inhibitor (Cki) Sic1 from Saccharomyces cerevisiae is a key regulator of the yeast cell cycle, which controls entrance into S phase and coordination between cell growth and proliferation. Its last 70 out of 284 residues display functional and structural homology to the inhibitory domain of mammalian p21 and p27. Sic1 has escaped systematic structural characterization until now. Here, complementary biophysical methods are applied to the study of conformational properties of pure Sic1 in solution. Based on sequence analysis, gel filtration, circular dichroism (CD), electrospray‐ionization mass spectrometry (ESI‐MS), and limited proteolysis, it can be concluded that the whole molecule exists in a highly disordered state and can, therefore, be classified as an IDP. However, the results of these experiments indicate, at the same time, that the protein displays some content in secondary and tertiary structure, having properties similar to those of molten globules or premolten globules. Proteolysis‐hypersensitive sites cluster at the N‐terminus and in the middle of the molecule, whereas the most structured region resides at the C‐terminus, including part of the inhibitory domain and the casein‐kinase‐2 (CK2) phosphorylation target S201. The mutations S201A and S201E, which are known to affect Sic1 function, do not have significant effects on the conformational properties of the pure protein. Proteins 2009;76:731–746.

Compaction Properties of an Intrinsically Disordered Protein: Sic1 and Its Kinase-Inhibitor Domain

IDPs in their unbound state can transiently acquire secondary and tertiary structure. Describing such intrinsic structure is important to understand the transition between free and bound state, leading to supramolecular complexes with physiological interactors. IDP structure is highly dynamic and, therefore, difficult to study by conventional techniques. This work focuses on conformational analysis of the KID fragment of the Sic1 protein, an IDP with a key regulatory role in the cell-cycle of Saccharomyces cerevisiae. FT-IR spectroscopy, ESI-MS, and IM measurements are used to capture dynamic and short-lived conformational states, probing both secondary and tertiary protein structure. The results indicate that the isolated Sic1 KID retains dynamic helical structure and populates collapsed states of different compactness. A metastable, highly compact species is detected. Comparison between the fragment and the full-length protein suggests that chain length is crucial to the stabilization of compact states of this IDP. The two proteins are compared by a length-independent compaction index.

Localization of lipase genes on Candida rugosa chromosomes

In the yeast Candida rugosa the lipase isozymes are encoded by a family of genes, five of which have been cloned and sequenced in our laboratory. In this paper we report on the identification and preliminary characterization of two new related sequences, thus extending this multigene family to seven members. The total DNA content of Candida cells was estimated by laser flow-cytometry at about 20 Mb. Eight chromosomes with sizes ranging between 100 kb and 2.1 Mb, as determined by comparison with S. cerevisiae chromosomal bands, were resolved by pulsed-field gel electrophoresis. The lipase-encoding genes were localized on chromosome I, therefore suggesting that they have originated through multiple duplication events of an ancestral gene.

Amplification of the CUP1 gene is associated with evolution of copper tolerance in Saccharomyces cerevisiae.

In living organisms, copper (Cu) contributes to essential functions but at high concentrations it may elicit toxic effects. Cu-tolerant yeast strains are of relevance for both biotechnological applications and studying physiological and molecular mechanisms involved in stress resistance. One way to obtain tolerant strains is to exploit experimental methods that rely on the principles of natural evolution (evolutionary engineering) and allow for the development of complex phenotypic traits. However, in most cases, the molecular and physiological basis of the phenotypic changes produced have not yet been unravelled. We investigated the determinants of Cu resistance in a Saccharomyces cerevisiae strain that was evolved to tolerate up to 2.5 g CuSO(4) l(-1) in the culture medium. We found that the content of intracellular Cu and the expression levels of several genes encoding proteins involved in Cu metabolism and oxidative stress response were similar in the Cu-tolerant (evolved) and the Cu-sensitive (non-evolved) strain. The major difference detected in the two strains was the copy number of the gene CUP1, which encodes a metallothionein. In evolved cells, a sevenfold amplification of CUP1 was observed, accounting for its strongly and steadily increased expression. Our results implicate CUP1 in protection of the evolved S. cerevisiae cells against Cu toxicity. In these cells, robustness towards Cu is stably inheritable and can be reproducibly selected by controlling environmental conditions. This finding corroborates the effectiveness of laboratory evolution of whole cells as a tool to develop microbial strains for biotechnological applications.

Electrospray-ionization mass spectrometry as a tool for fast screening of protein structural properties

Since the early 1990s, electrospray‐ionization mass spectrometry (ESI‐MS) has encountered growing interest as a complementary tool to established biochemical and biophysical methods for investigating protein structure and conformation. Nowadays, applications of ESI‐MS to protein investigation span from the area of analytical biochemistry to that of structural biology. This review focuses on applications of this technique to the analysis of protein conformational properties and molecular interactions, underscoring their possible relevance for molecular biotechnology, although representing a still very young field. An introductive section presents the major issues related to theoretical and technical aspects of ESI‐MS under non‐denaturing conditions. Examples from our work and from the literature illustrate which kind of information can be obtained concerning key issues in biotechnology such as stability and aggregation of proteins under both near‐native and challenging conditions, and interactions with other proteins, ligands and cofactors.