Structural features of interferon-gamma aggregation revealed by hydrogen exchange

Using hydrogen-deuterium exchange (HX) and electrospray ionization mass spectrometry, we have investigated the stability and structural changes of recombinant human interferon-gamma (IFN-gamma) during aggregation induced by guanidine hydrochloride (GdnHCl) and potassium thiocyanate. First, HX labeling was initiated after the amorphous aggregates were formed to probe the tertiary structure of the aggregated state. Second, labeling was performed at low protein concentrations to assess stability under aggregation prone conditions. In 1 M GdnHCl, the stability of IFN-gamma was greatly reduced and much less protection from HX in solution was observed. Exchange under these conditions was slower in helix C than in the rest of the protein. Aggregates formed in 1 M GdnHCl showed a HX pattern consistent with a partially unfolded state with an intact helix C. Although aggregates formed in 0.3 M KSCN exhibited a HX pattern similar to those formed in GdnHCl, the solution phase HX pattern in 0.3 M KSCN was surprisingly comparable to that of the native state. Varying the aggregation time before performing HX revealed that KSCN first precipitated native protein and then facilitated partial unfolding of the precipitated protein. These results show that helix C, which forms the hydrophobic core of the IFN-gamma dimer, is highly protected from HX under native conditions, is more stable in GdnHCl than the rest of the protein and remains intact in both GdnHCl- and KSCN-induced aggregates. This suggests that native-state HX patterns may presage regions of the protein susceptible to unfolding during aggregation.     

Relations between fibrinogen degradation products and heparinocytes.

In the emergency reaction there is a short-time increase of the values of basophilic leukocytes in connection with normal values of fibrinogen degradation products (FDP). After diminuation of basophils FDP are to be detected. In the chronical ill and under different hormonal contraceptives these two processes are overlapped disturbing a unique negative or positive correlation of heparinocytes and FDP. Concerning the situation under standardized conditions or in single cases worthful conclusions are possible about compensation or decompensation of a latent disseminated intravascular coagulation.     

Conformational differences between arrestin2 and pre-activated mutants as revealed by hydrogen exchange mass spectrometry

Arrestins are regulatory proteins that bind specifically to ligand-activated phosphorylated G protein-coupled receptors to terminate G protein-mediated signaling, cause the internalization of the receptor-arrestin complex, and initiate additional intracellular signaling cascades. Multiple lines of evidence suggest that arrestin normally exists in an inactive basal state and undergoes conformational activation in the process of receptor binding. "Pre-activated" phosphorylation-independent arrestin mutants display increased binding to ligand-activated but unphosphorylated receptors. The mutations are believed to expose key receptor-binding regions, allowing the mutants to mimic, to some extent, the transition of arrestin to its active state. In the present study, amide hydrogen exchange (HX) and mass spectrometry (MS) were used to examine the inactive conformation of wild-type arrestin2 and compare its solution conformation with two pre-activated mutants (R169E and 3A (I385A, V386A, F387A)). The results suggest an unexpected level of structural organization within arrestin elements containing clathrin and adaptin2-binding sites that were previously believed to be completely disordered. Increased deuterium incorporation was observed in both mutant forms compared with wild-type, indicating a change in the conformation of the mutants. Three regions demonstrated significant differences in deuterium incorporation: the first 33 residues of the N terminus and residues 243-255 (both previously implicated in receptor interaction), and residues 271-299. The results suggest that subtle differences in conformation are responsible for the significant difference in biological activity displayed by pre-activated arrestin mutants and that similar changes occur in the process of arrestin binding to the receptor.     

Characterization of the terminal degradation products of canine fibrinogen by plasmin.

Fibrinogen, isolated from canine plasma by the successive procedures of (1) freezing and thawing, (2) fractional precipitation with 25% saturated (HN4)2SO4 and (3) Sepharose 6B gel-filtration, had a molecular weight of 282 000 by the rapid sedimentation equilibrium method. However, a molecular weight for canine fibrinogen of 332 000, which is closer to that reported for human and bovine fibrinogens (340 000 plus or minus 20 000), was obtained from the sum of the molecular weights of the Aalpha, Bbeta and gamma chains, determined from dodecylsulfate gel electrophoretic patterns of reduced fibrinogen. Canine fibrinogen, subjected to proteolysis by urokinase-activated plasminogen for 24 h, contained degradation fragments D and E which were isolated by starch block electrophoresis and Sephadex G-200 gel-filtration. The purified D and E fragments with sedimentation coefficients of 5.0 S and 2.5 S had weight average molecular weights of 89 000 and 42 000, respectively by the rapid sedimentation equilibrium method. The ratio of D to E was 2:1 per parent fibrinogen molecule. Antigenic analysis according to anti-fibrinogen antiserum showed that both D and E fragments were antigenically deficient to native fibrinogen and revealed a reaction of non-identity with each other. Upon immunoelectrophoresis at pH 8.2, D and E had different electrophoretic mobilities. Preliminary studies indicate that based on thrombin time alone, D has anticoagulant activity while E appears to be a coagulation potentiator. Canine fibrinogen apparently consist of two core fragments with dissimilar chemical characteristics in common with the fundamental structures of human and bovine fibrinogens.     

The role of ligand-ligand interactions in competition by fibrinogen and fibrin degradation products for fibrinogen binding to human platelets

Binding to human platelets of radioiodinated human fibrinogen and fragments X, Y, D, D₁ dimer and E was studied to determine the domain of the fibrinogen molecule responsible for binding to the platelet receptor. Although the fragments did not bind, some wer able to complete for the binding of fibrinogen to platelets. It was postulated that the fragments bound to fibrinogen and subsequently interfered with its binding to the receptor. Two approaches were developed to test this hypothesis. In the first technique, molecular exclusion on Sephacryl S-200 superfine was utilized to examine the interaction of radiolabeled fragments with fibrinogen. In the second seties of studies, fibrinogen-Sepharose was prepared and the binding of degradation products directly determined. A spin dialysis apparatus was employed in each case to achieve rapid separation of bound and free radioligand. These studies demonstrated that fragments D and E bind to fibrinogen. Therefore, the mechanism by which degradation products interfere with fibrinogen binding to the platelet receptor is ligand-ligand interaction rather than binding of the fragments to the receptor. Since none of the radiolabeled degradation products bound to platelets, it appears that receptor recognition requires the intact molecule.     

Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry

Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.     

Protein conformation in amorphous solids by FTIR and by hydrogen/deuterium exchange with mass spectrometry

Solid-state hydrogen/deuterium exchange (ssHDX) with electrospray ionization mass spectrometry (ESI-MS) and Fourier transform infrared (FTIR) spectroscopy were used to assess protein conformation in amorphous solids. Myoglobin, lysozyme, β-lactoglobulin, ribonuclease A, E-cadherin 5, and concanavalin A were co-lyophilized with carbohydrates (trehalose, raffinose, and dextran 5000), linear polymers (polyvinyl alcohol and polyvinyl pyrrolidone) or guanidine hydrochloride (negative control). For ssHDX, samples were exposed to D₂O vapor at 33% relative humidity and room temperature, and then reconstituted at low temperature (4°C) and pH 2.5 and analyzed by ESI-MS. Peptic digestion of selected proteins was used to provide region-specific information on exchange. FTIR spectra were acquired using attenuated total reflectance. FTIR and ssHDX of intact proteins showed preservation of structure by raffinose and trehalose, as indicated by FTIR band intensity and protection from exchange. ssHDX of peptic digests further indicated that these protective effects were not exerted uniformly along the protein sequence but were observed primarily in α-helical regions, a level of structural resolution not afforded by FTIR. The results thus demonstrate the utility of HDX with ESI-MS for analyzing protein conformation in amorphous solid samples.     

Origin of urinary fibrin/fibrinogen degradation products in glomerulonephritis.

To elucidate the origin of the fibrin/fibrinogen degradation products (F.D.P.) occurring in the urine in glomerulonephritis 28 patients with glomerulonephritis were examined for renal fibrinolytic activity, F.D.P. in urine and serum, and blood fibrinolytic activators and blood fibrinolytic activators and inhibitors. Unlike the glomerful of healthy kidneys, which were fibrinolyticly inactive, those of kidneys with glomerulonephritis constantly showed fibrinolytic activity. The presence or absence of fibrin in the glomeruli was almost always accompanied by, respectively, the presence or absence of urinary F.D.P., which suggested a renal origin of urinary F.D.P. in glomerulonephritis. The low fibrinolytic activity of the blood and the absence of F.D.P. in the serum of these patients make it unlikely that the urinary F.D.P. in glomerulonephritis result from glomerular filtration.     

On the separation of fibrinogen degradation products D and E.

Separation of fibrinogen degradation products D and E by means of gel chromatography cannot be achieved at neutral pH even in the presence of high ionic strength of the elution buffer. It is assumed that fragments D and E are linked together in a complex preventing the separation despite different molecular weights of both components. By means of addition of chaotropic substances like 1 M Kl to the elution buffer clear separation of degradation products D and E on Sephadex G-200 columns can be achieved.     

Cores and pH-dependent dynamics of ferredoxin-NADP+ reductase revealed by hydrogen/deuterium exchange

NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP(+) reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP(+))-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD(r) values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD(r) values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP(+)-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP(+)-binding domain at pD(r) 8.0, the daytime pH in chloroplasts, than at pD(r) 6.0 is likely to be involved in the increased binding of NADP(+) for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.