11 research outputs found
Native state dynamics affects the folding transition of porcine pancreatic phospholipase A2.
Porcine pancreatic phospholipase A2, a small and disulfide rich protein, is extremely resistant against chemically or thermally induced unfolding. Despite this marked resistance, the protein displays broad unfolding transitions resulting in comparatively low apparent thermodynamic stability. Broad unfolding transitions may result from undetected folding intermediates, residual structures in the unfolded state or an inhomogeneity of the native state. Using circular dichroism, fluorescence, and NMR spectroscopy, we ruled out the existence of stably populated folding intermediates, whereas UV absorbance measurements hinted at stable residual structures in the unfolded state. These residual structures proved, however, to have no impact on the folding parameters. Studies by limited proteolysis, CD, and NMR spectroscopy under non-denaturing conditions suggested pronounced dynamics of the protein in the native state, which as long as unrestrained by acidic pH or bound Ca(2+) ions exert considerable influence on the unfolding transition
Conformational Shift of a Ī²āHairpin Peptide upon Complex Formation with an Oligoāproline Peptide Studied by Mass Spectrometry
So-called super-secondary structures such as the beta-hairpin, studied here, form an intermediate hierarchy between secondary and tertiary structures of proteins. Their sequence-derived 'pure' peptide backbone conformation is combined with 'remote' interstrand or interresidue contacts reminiscent of the 3D-structure of full-length proteins. This renders them ideally suited for studying potential nucleation sites of protein folding reactions as well as intermolecular interactions. But beta-hairpins do not merely serve as model systems; their unique structure characteristics warrant a central role in structural studies on their own. In this study we applied photo cross-linking in combination with high-resolution mass spectrometry and computational modeling as well as with ion mobility-mass spectrometry to elucidate these structural properties. Using variants of a known beta-hairpin representative, the so-called trpzip peptide and its ligands, we found evidence for a conformational transition of the beta-hairpin and its impact on ligand binding.113sciescopu
NMR Experiments Provide Insights into Ligand-Binding to the SARS-CoV-2 Spike Protein Receptor-Binding Domain
We have used chemical shift perturbation (CSP) and saturation transfer difference (STD) NMR experiments to identify and characterize the binding of selected ligands to the receptor-binding domain (RBD) of the spike glycoprotein (S-protein) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We also subjected full-length S-protein to STD NMR experiments, allowing correlations with RBD-based results. CSPs reveal the binding sites for heparin and fondaparinux, and affinities were measured using CSP titrations. We then show that -2,3-sialyllactose binds to the S-protein but not to the RBD. Finally, combined CSP and STD NMR experiments show that lifitegrast, a compound used for the treatment of dry eye, binds to the linoleic acid (LA) binding pocket with a dissociation constant in the M range. This is an interesting finding, as lifitegrast lends itself well as a blueprint for medicinal chemistry, eventually furnishing novel entry inhibitors targeting the highly conserved LA binding site
Structural models for laminin Ī³1 L4.
<p>Shown are the two best-scoring models generated by comparative modeling based on 13 structural homologues that have been identified by fold recognition. The residues are colored according to their Rosetta total score. Scores below zero (yellow-green color) indicate energetically favorable conformations.</p
Contacts of laminin Ī³1 LEb2ā4 wild type with nidogen-1.
<p>The LEb2ā4 structure (red) is taken from PDB entry 1NPE. Nidogen-1 (grey) is schematically depicted as a combination of the crystal structures 1GL4 (G2 domain) and 1NPE (G3 domain) and representative models of the remaining domains. Residues involved in intermolecular contacts are shown as spheres. Gray dotted lines represent verified cross-links.</p
Overview of cross-links within the nidogen-1 G3/laminin Ī³1 LEb2ā4 complex.
<p>CĪ±āCĪ± distances of cross-linked residues were determined for the unmodified crystal structure (PDB entry 1NPE) as well as for the Rosetta models with the best Rosetta total score and atom-pair constraint score, respectively (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112886#pone-0112886-g006" target="_blank">Figure 6</a>). For intermolecular contacts, residues are assigned to the respective protein. All other cross-links are located within nidogen-1.</p><p>Overview of cross-links within the nidogen-1 G3/laminin Ī³1 LEb2ā4 complex.</p
Diagonal plots of all cross-links identified.
<p>Cross-links are assigned to domains based on the UniProt KB entry P10493 (nidogen-1) and the laminin nomenclature of Aumailley <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112886#pone.0112886-Aumailley1" target="_blank">[5]</a>. Unannotated areas within the sequences are named āuaā. Corresponding to the number of inter-domain contacts, areas of intersection are color-coded from white (none) to dark grey (maximum). (A) Intramolecular cross-links within nidogen-1. The globular domains G1, G2, and G3 are denoted by dotted lines. Cross-links located nearby the diagonal border represent contacts between domains being close to each other in the protein sequence. (B) Cross-links between nidogen-1 and laminin Ī³1 wild type (upper panel) as well as N836D variants (lower panel). The LEb3 domain, bearing the N836D mutation, is marked with an asterisk.</p
Refined models of the nidogen-1 G3/laminin Ī³1 LEb2ā4 complex.
<p>Based on PDB entry 1NPE and the identified cross-links, modified structural models for the high-affinity interaction region of laminin Ī³1 and nidogen-1 were generated. Cross-linked residues are displayed as spheres. CĪ±āCĪ± distances are given in Ć
. (A) Model with the best Rosetta total score and a Rosetta atom-pair constraint score ranking among the top 2.5%. (B) Model with the best Rosetta atom-pair constraint score and a Rosetta total score ranking among the top 2.5%. (C) Alignment of both models and the unmodified crystal structure 1NPE (black). The orientation of LEb2ā4 clearly has changed during structural refinement. The Ī²-propeller fold of the G3 domain is still intact.</p
Arrangement of (A) nidogen-1 and (B) laminin Ī³1 short arm.
<p>The domains are color-coded with respect to the availability of crystal structures (green), template structures for comparative modeling (yellow) or neither of both (red). PDB IDs of the respective crystal structures are indicated in italics. (A) Nidogen-1 domain assignments are according to the UniProt KB entry P10493. Additionally, the historic domain names (G1ālinkāG2ārodāG3) are given. (B) Laminin domain designations follow the nomenclature of Aumailley <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112886#pone.0112886-Aumailley1" target="_blank">[5]</a>. Laminin Ī³1 short arm is part of the heterotrimeric protein laminin-111, the overall structure of which is schematically depicted.</p
<i>De novo</i> folded models of nidogen-1 G1.
<p>The ten best-scoring structures among the final models were all derived from four initial centroid models of the G1 domain. Structures representing three of these centroid models are shown here. These models comply with the single distance constraint in this region that was identified by cross-linking/MS. Cross-linked residues are displayed as black sticks. CĪ±āCĪ± distances are given in Ć
. The residues are colored according to their Rosetta total score. Scores below zero (yellow-green color) indicate energetically favorable conformations. The identifiers of the underlying centroid models are indicated.</p