Influence of carbohydrate on structure, stability, and function of gelatin-binding fragments of fibronectin.

The gelatin-binding region of fibronectin contains three Asn-linked carbohydrate moieties, one on the second type II module and two on the eighth type I "finger" module. Carbohydrate groups were enzymatically removed from two nonoverlapping gelatin-binding fragments (GBFs), 21-kDa GBF (modular composition I8-I9) and, with much greater difficulty, 30-kDa GBF (modular composition I6-II1-II2-I7). The gelatin-binding properties of these fragments were affected only slightly or not at all. Fluorescence and calorimetric analyses indicated that module I8 was strongly destabilized by deglycosylation such that the apo form melts near physiological temperature. A similar effect was caused by decreasing the pH of the holo form to 6.0, suggesting that one or more histidines are important for stability of module I8. The 21-kDa fragment exhibited an acid-induced change in fluorescence that occurred at higher pH in the deglycosylated derivative, providing further evidence of a stabilizing role for one or both carbohydrate moieties. By contrast, the stability of module II2 was unaffected by removal of its single carbohydrate. The effects of carbohydrate on the stability of module I8 may be important in efforts to express it or fragments containing it in bacteria.

Domain structure of the Fib-1 and Fib-2 regions of human fibronectin. Thermodynamic properties of the type I finger module.

The stability of the Fib-1 (29 kDa) and Fib-2 (19 kDa) fragments of human fibronectin as well as several different subfragments and isolated type I "finger" modules were studied under various solvent conditions by differential scanning calorimetry and fluorescence spectroscopy. It was established that all fibronectin fingers constitute independently folded domains whose melting temperatures range from 54 to 108 degrees C. The difference between heat capacities of the native and denatured states (delta Cp) is low, about 0.03 cal/K-g, which is consistent with the relatively low percentage of hydrophobic amino acids and the consequent small change in non-polar surface area exposed to the solvent upon denaturation. The free energy of unfolding at 25 degrees, as calculated from the calorimetric data or measured directly by titration with GdmSCN is also small, in the range of 2.4 to 6.7 kcal/mol. The small delta G value and its flat dependence on temperature (determined by delta Cp) translates the small variations in delta G between fingers into large variations in tm. The small value of delta G also indicates that finger modules are structurally rather fragile which may account for their sensitivity to proteolysis; almost any cleavage within either of the two disulfide loops destroys the cooperative structure and abolishes the corresponding melting transition. The fact that some fingers exhibit large decreases in tm upon separation from more stable neighbors with which they interact can also be viewed as a consequence of the low values of delta G and delta Cp.

Effect of tethered peptidylchloromethylketone inhibitors on thermal stability and domain interactions of urokinase and other serine proteases.

The melting of several serine proteases that had been reacted with different peptidylchloromethylketone (cmk) inhibitors was studied by fluorescence spectroscopy and calorimetry. These inhibitors, which cross-link the two domains of the proteases, invariably increased the melting temperature by as much as 28.5 degrees C. The magnitude of the effect was dependent on the size and composition of the peptide moieties. The delta G of unfolding of tosyl-Phe-cmk-chymotrypsin was 13.5 kcal/mol compared to only 8.3 kcal/mol for chymotrypsin. Binding of cmk inhibitors also protected the two interacting domains of urokinase from acid-induced decooperation and caused them to merge into a highly cooperative structure upon refolding at low pH. Fluorescence-detected melting curves of Glu-Gly-Arg-cmk-urokinase indicated that unfolding/refolding at pH 4.5 is characterized by dramatic hysteresis; the cooling curves fell close to those obtained upon heating or cooling of the uninhibited enzyme. Upon second heating, the melting curves were similar to those of the original. The hysteresis effects are interpreted as follows. The tethered tripeptide binds to the active site, causing the protein to melt at much higher temperature in a single cooperative step, as if the two domains are merged into one cooperative unit. Upon cooling, the unfolded protein, with the inhibitor still attached, refolds at the same temperature as the underivatized protein. Only after the native structure is formed does the peptide moiety again bind and stabilize toward a second heating. At lower pH, second heating produced biphasic or triphasic melting curves that were attributed to differential protonation of acid-titratable groups on the enzyme and/or inhibitor at the time of refolding. Similar effects were observed with other trypsin-like proteases, indicating that the hysteresis and bi- and triphasic refolding at low pH are rather general for this class of enzyme.

Reversible unfolding of an isolated heparin and DNA binding fragment, the first type III module from fibronectin.

Several fragments containing all or part of the first type III homology unit of fibronectin were isolated and their folding properties examined by fluorescence spectroscopy and differential scanning calorimetry. Each fragment exhibits a reversible unfolding transition when heated or titrated with guanidinium chloride. This indicates that an isolated type III module can fold independently in the absence of neighboring modules. A comparison of the specific enthalpies of unfolding of these fragments with those of well-studied globular proteins suggests that this type III unit is composed of a stable core flanked by less compact or unstructured regions. Comparison of the heparin-binding properties of these fragments revealed that removal of 12 amino acids from the amino terminus of the largest one (Ile-585 to Val-675) increased its affinity for immobilized heparin such that it now binds at physiological ionic strength.

Domain structure and domain-domain interactions of recombinant tissue plasminogen activator.

The melting of recombinant tissue plasminogen activator (rtPA) has been investigated by differential scanning calorimetry and fluorescence spectroscopy. At neutral pH, rtPA melts with only partial reversibility in a single sharp peak that can be deconvoluted into four transitions. By contrast, at acidic pH the melting process is spread over a broad range of temperature and is highly reversible. Under these conditions five transitions are resolved by deconvolution analysis. Additional measurements in 6 M guanidinium chloride reveal a sixth transition representing an extremely stable domain. Comparison of the melting curves of several fragments with those of the parent protein allowed all of the transitions to be assigned. The results indicate that rtPA is comprised of six independently folded domains. Two of these domains correspond to the two kringle modules whose thermodynamic properties are similar to those of the kringles in plasminogen. Two additional domains are formed by the epidermal growth factor (EGF)-like and finger modules, the latter of which is extremely stable, requiring the presence of a chemical denaturant for its melting to be observed. The serine protease module contains two more domains which at neutral pH melt cooperatively in a single transition but at low pH melt independently, accounting for the greater number of transitions observed there. Measurements with a 50-kDa fragment lacking the C-terminal half of the serine protease module and with a variant lacking the finger and EGF domains indicate that the serine protease domains interact strongly with and are stabilized by the finger and/or EGF domains in the intact protein. This interaction between domains located at opposite ends of the rtPA molecule produces a more compact structure. A better understanding of such interactions may enhance efforts to engineer plasminogen activators with improved thrombolytic properties.

Fluorescence spectroscopic analysis of ligand binding to kringle 1 + 2 + 3 and kringle 1 fragments from human plasminogen.

The ligand binding of kringle 1 + 2 + 3 and kringle 1 from human plasminogen has been investigated by fluorescence spectroscopy. Analysis of fluorescence titration of kringle 1 + 2 + 3 with 6-aminohexanoic acid shows that this fragment, besides the high-affinity lysine-binding site with Kd = 2.9 microM, contains two additional lysine-binding sites which differ in binding strength (Kd = 28 microM and Kd = 220 microM). This strongly suggests the existence of a lysine-binding site in each of the first three kringles. 6-Aminohexanoic acid, pentylamine, pentanoic acid and arginine were used for investigation of the ligand specificity of isolated kringle 1 prepared by pepsin hydrolysis of kringle 1 + 2 + 3. It has been established that kringle 1 has high affinity to 6-aminohexanoicacid, pentylamine and arginine (Kd values are 3.2 microM, 4.8 microM and 4.3 microM, respectively). At the same time pentanoic acid did not bind with kringle 1. These facts indicate, firstly, a broad ligand specificity of kringle 1 and, secondly, the paramount importance of the positively charged group of the ligand for its interaction with lysine-binding site of this kringle.

Analysis of ligand binding to kringles 4 and 5 fragments from human plasminogen.

The interaction of the isolated kringles 4 and 5 from human plasminogen with 6-aminohexanoic acid, pentylamine, pentanoic acid and arginine has been quantitatively characterized by scanning calorimetry and fluorescent spectroscopy. It has been found that the ligands with the positively charged group have a good binding ability while pentanoic acid in comparison with 6-aminohexanoic acid being devoid of amino group does not interact with the kringles under study. The positively charged group of the ligand is suggested to play a crucial role in ligand binding with the lysine-binding site.

Domains in human plasminogen.

Calorimetric studies of intramolecular melting of human plasminogen and of its fragments under various solvent conditions show that the intact plasminogen molecule consists of seven compact co-operative subunits, which can be regarded as structural domains. Five of these domains are formed by the homologous regions, the kringles, two domains are formed by the C-terminal part of the polypeptide chain that is split at activation, forming the light chain in plasmin, while the initial 76 amino acid residue peptide does not form any compact co-operative structure. The specific influence of epsilon-aminocaproic acid on the stability of the first, the fourth and, to a lesser extent, on the second kringle domain, provides evidence that these three domains in plasminogen possess lysine-binding ability. The first four kringle domains are almost independent in the molecule, while the fifth interacts with that part of the light chain not included in either of the two domains of this chain. These two domains are of different size and co-operate strongly in plasminogen, but at its activation into plasmin they decooperate and the stability of the smaller domain, which is formed by the N-terminal part of the light chain, decreases significantly. Since the light chain is responsible for the proteolytic activity of plasmin, it becomes clear that the active site of this protein is composed of two domains, as is the case for other serine proteases.