No abstract

To understand how proteins fold in vivo, it is important to investigate the effects of macromolecular crowding on protein folding. Here, the influence of crowding on in vitro apoflavodoxin folding, which involves a relatively stable off-pathway intermediate with molten globule characteristics, is reported. To mimic crowded conditions in cells, dextran 20 at 30% (w/v) is used, and its effects are measured by a diverse combination of optical spectroscopic techniques. Fluorescence correlation spectroscopy shows that unfolded apoflavodoxin has a hydrodynamic radius of 37 ؎ 3 Å at 3 M guanidine hydrochloride. Förster resonance energy transfer measurements reveal that subsequent addition of dextran 20 leads to a decrease in protein volume of about 29%, which corresponds to an increase in protein stability of maximally 1.1 kcal mol ؊1 . The compaction observed is accompanied by increased secondary structure, as far-UV CD spectroscopy shows. Due to the addition of crowding agent, the midpoint of thermal unfolding of native apoflavodoxin rises by 2.9°C. Although the stabilization observed is rather limited, concomitant compaction of unfolded apoflavodoxin restricts the conformational space sampled by the unfolded state, and this could affect kinetic folding of apoflavodoxin. Most importantly, crowding causes severe aggregation of the off-pathway folding intermediate during apoflavodoxin folding in vitro. However, apoflavodoxin can be over expressed in the cytoplasm of Escherichia coli, where it efficiently folds to its functional native form at high yield without noticeable problems. Apparently, in the cell, apoflavodoxin requires the help of chaperones like Trigger Factor and the DnaK system for efficient folding.

The regulation of cell polarity plays an important role in chemotaxis. Previously, two proteins termed GbpC and GbpD were identified in Dictyostelium, which contain RasGEF and cyclic nucleotide binding domains. Here we show that gbpC-null cells display strongly reduced chemotaxis, because they are unable to polarise effectively in a chemotactic gradient. However, gbpD-null mutants exhibit the opposite phenotype: cells display improved chemotaxis and appear hyperpolar, because cells make very few lateral pseudopodia, whereas the leading edge is continuously remodelled. Overexpression of GbpD protein results in severely reduced chemotaxis. Cells extend many bifurcated and lateral pseudopodia, resulting in the absence of a leading edge. Furthermore, cells are flat and adhesive owing to an increased number of substrate-attached pseudopodia. This GbpD phenotype is not dependent on intracellular cGMP or cAMP, like its mammalian homolog PDZ-GEF. Previously we showed that GbpC is a high-affinity cGMP-binding protein that acts via myosin II. We conclude that cGMP activates GbpC, mediating the chemoattractant-induced establishment of cell polarity through myosin. GbpD induces the formation of substrate-attached pseudopodia, resulting in increased attachment and suppression of polarity.

Autoactivation of FXII by polyP, of the size found in platelets, proceeds via an active single-chain intermediate. scFXII-polyP70 shows activity towards physiological substrates, and may represent the primary event in initiating contact activation in vivo.

Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N- and O-glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O-glycosylated N-terminal region. Five novel and five known O-glycosylation sites were identified, carrying mainly core1-type O-glycans. In addition, we detected a heavily O-glycosylated portion spanning from Thr82-Ser121 with up to 16 O-glycans attached. Likewise, all known six N-glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N-glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications.