The effects of hydrostatic pressure on the conformation of plasminogen

Abstract

Plasminogen undergoes a large conformational change when it binds 6-aminohexanoate. Using ultraviolet absorption spectroscopy and native PAGE, we show that hydrostatic pressure brings about the same conformational change. The volume change for this conformational change is 233 mL´mol 21 . Binding of ligand and hydrostatic pressure both cause the protein to open up to expose surfaces that had previously been buried in the interior.Keywords: conformational changes; hydrostatic pressure; plasminogen.Plasminogen, a glycoprotein present in vertebrate blood, carries out no known reactions. It is the inactive proenzyme form of plasmin, a protease of the chymotrypsin family. As a proenzyme, it is capable of binding to macromolecular or supramolecular surfaces which contain exposed carboxyterminal lysines or some exposed amino sugars. Having bound to a surface, the proenzyme undergoes a conformational change and can then be activated by any one of several proteases or other plasminogen molecules complexed to the same surface. Activation of the proenzyme involves cleavage of one peptide bond; the two chains remain joined by disulfide bonds [1]. The activated enzyme, plasmin, is one of the enzymes that cleaves fibrin. Native plasminogen is called gluplasminogen [2]. It is converted to several other plasminogen forms, termed lys-plasminogen, by cleavage of the amino terminus. The native structure contains < 790 amino acids [3]; it consists of an N-terminal domain, five kringles and a C-terminal proteolytic domain. The activation cleavage that gives rise to plasmin, occurs in the linker region between kringle 5 and the proteolytic domain. The secondary structure of plasminogen is interesting: it includes the five kringles, all of which are quite similar; each contains < 80 amino acids packed as four pairs of antiparallel b-sheets with b-linkers as shown by far UV CD [4], and confirmed by both X-ray crystallography [5,6] and solution NMR of isolated kringles [7]. The name for this folding pattern derives from the Danish word for pretzel. The protease domain has the same tertiary structure as that of the pancreatic serine proteases [8]. Conformational changes occur when plasminogen binds C-terminal lysine derivatives or analogous compounds, such as 6-aminohexanoate (6-AH). It is believed that the binding of these small molecules mimics the physiological binding of macromolecular surfaces such as that of the fibrin clot. At least three 6-AH can bind. Binding to tightest site (kringle 1) does not cause a detectable conformational change [9] but binding to kringles 4 and 5 results in major structural changes [10] for glu-plasminogen and minor changes for the lys-plasminogens. Small angle neutron scattering indicated that when glu-plasminogen binds 6-AH the protein changes from a prolate ellipsoid with a radius of gyration of 39 A Ê and maximum dimension of 147 A Ê to one with radius of gyration of 56 A Ê and maximum dimension of < 250 A Ê [4]. Low angle X-ray scattering showed a more compact structure for the unligated form [11] ...

Protein Corona in Response to Flow: Effect on Protein Concentration and Structure

“…Because much of the flow-dependent research is driven by bioprocessing and storage of therapeutic proteins (47), the structural changes of plasminogen in response to flow have not been examined previously. However, structural changes of plasminogen have been examined in response to 6-aminohexanoate and hydrostatic pressure (38,39,51,52). This previous work suggests that the resulting structural changes mimic the structural changes that result from plasminogen binding to surfaces, such as fibrin clots, which is the initial step in activation (51,52).…”

“…However, structural changes of plasminogen have been examined in response to 6-aminohexanoate and hydrostatic pressure (38,39,51,52). This previous work suggests that the resulting structural changes mimic the structural changes that result from plasminogen binding to surfaces, such as fibrin clots, which is the initial step in activation (51,52). In this case, it is possible that small structural changes due to flow aid in the activation of plasminogen.…”

The Interaction of Canine Plasminogen with Streptococcus pyogenes Enolase: They Bind to One Another but What Is the Nature of the Structures Involved?

“…The preparation of dPgn has been described [58]. Our standard assay for dPgn activation to plasmin consists of incubating dPgn with trypsin (40 nM), monitoring enzymatic activity using Spectrozyme and monitoring the change in gel pattern as the dPgn converts first to lys-dPgn and then to plasmin.…”

Inactivation of creatine kinase by high pressure may precede dimer dissociation

“…The significant advantage is that upon pressure release no chemical residues are present in the solution and the changes can be analyzed in real time. Pressure is widely used to follow the dissociation of oligomeric proteins [10–14], and to investigate protein conformational changes or denaturation [15–17]. The CK reaction was used 15 years ago as a model reaction to investigate the pressure effects on an activated complex [18].…”