NMR Solution Structure of the 205−316 C-Terminal Fragment of Thermolysin. An Example of Dimerization Coupled to Partial Unfolding

Abstract

The solution structure of the C-terminal fragment 205-316 of thermolysin has been determined by 1 H-NMR methods. The fragment forms a dimer in which each subunit has two different regions: the largely disordered N-terminal segment 205-260 and the structurally well-defined segment 261-316. The structured part of each subunit is composed of three helices and is largely coincident with the corresponding region in the solution structure of the dimer formed by the shorter fragment 255-316, which in turn coincides with the crystallographic structure of intact thermolysin. As with the fragment 255-316, the subunit interface is highly hydrophobic and coincides topologically with the one between the segment 255-316 and the rest of the protein in the intact enzyme. A fourth helix (residues 235-246), present in the segment 205-316 of native thermolysin, is mostly disordered in the dimer formed by the fragment 205-316. The location of the fourth helix in the native structure of intact thermolysin does not allow the formation of the dimer interface observed in the solution structure of the fragment 255-316. Under the NMR conditions, dimer formation is energetically more favorable than the dissociated monomers. The latter, based on calorimetric data, was proposed to have partial structure in the region 205-254 as in native thermolysin. Thus, it appears that the assembly of the dimer would require an initial unfolding in the region 205-254 of the monomer.

Local Unfolding Is Required for the Site-Specific Protein Modification by Transglutaminase

“…26 NMR measurements on 205− 316Th confirmed the disordered N-terminal region (approximately residues 205−261) and a folded and nativelike helical structure in the C-terminal region. 51,52 Overall, the results of the derivatization of Gln and Lys residues of 205−316Th with TGase correlate with those of limited proteolysis, because TGase and proteases attack the fragment substrate at its disordered N-terminal region (see Figure 4B).…”

“…The C-terminal fragment 205/206–316 is capable of folding into a nativelike structure in a manner independent of the rest of the polypeptide chain, thus possessing protein domain properties. − Here, we explored the TGase modification at both Gln and Lys residues of fragment 205–316, containing in its amino acid sequence seven Gln and seven Lys residues (Figure S1 and Table S1 of the Supporting Information). Analysis by RP-HPLC and ESI-MS of the reaction mixture with DC after reaction for 6 h showed that the N-terminal Met can be partly removed during the reaction (Figure A), as shown by the ESI-MS data (Table S7 of the Supporting Information).…”

“…(A) RP-HPLC analyses of the TGase-mediated derivatives of 205− 316Th. A dashed line and a straight line indicate the chromatograms of the native thermolysin fragment and of the DC-and ZQG-conjugated species, respectively, after reaction for 6 h. (B) Scheme of the secondary structure of 205−316Th 51,52. The three helices (H1−H3) are depicted as boxes.…”

An ambiguity principle for assigning protein structural domains

“…3U, in salmon) ( 24 ), whereas its alternative delimits a domain that has been shown to be able to fold partially (Fig. 3U, in green cyan) ( 25 ). Another example is α-lactalbumin (PDB: 1a4v), which is annotated as a one-domain protein in CATH, SCOP, ECOD, and Pfam, whereas our algorithm isolates an α-helical domain in its best alternative assignment (Fig.…”

Observation of a Pressure-Induced Unfolding Intermediate of Thermolysin by Using Pressure-Jump Method

“…3 These domains seem to have differential stability against physical and chemical perturbations and, in addition, certain partial sequences of these domains are known to fold autonomously under moderate conditions. [7][8][9][10] We have reported the effects of high pressure on TLN activity and spectroscopic properties over a range of temperature and pressure. [4][5][6] The pressure-induced spectroscopic changes of TLN were explained by a simple two-state transition model, accompanied with a large and negative reaction volume change, ∆V.…”