Denaturant-dependent conformational changes in a beta-trefoil protein: global and residue-specific aspects of an equilibrium denaturation process.
Conformational properties of the folded and unfolded ensembles of human interleukin-1 receptor antagonist (IL-1ra) are strongly denaturant-dependent as evidenced by high-resolution two-dimensional nuclear magnetic resonance (NMR), limited proteolysis, and small-angle X-ray scattering (SAXS). The folded ensemble was characterized in detail in the presence of different urea concentrations by (1)H-(15)N HSQC NMR. The beta-trefoil fold characteristic of native IL-1ra was preserved until the unfolding transition region beginning at 4 M urea. At the same time, a subset of native resonances disappeared gradually starting at low denaturant concentrations, indicating noncooperative changes in the folded state. Additional evidence of structural perturbations came from the chemical shift analysis, nonuniform and bell-shaped peak intensity profiles, and limited proteolysis. In particular, the following nearby regions of the tertiary structure became progressively destabilized with increasing urea concentrations: the beta-hairpin interface of trefoils 1 and 2 and the H2a-H2 helical region. These regions underwent small-scale perturbations within the native baseline region in the absence of populated molten globule-like states. Similar regions were affected by elevated temperatures known to induce irreversible aggregation of IL-1ra. Further evidence of structural transitions invoking near-native conformations came from an optical spectroscopy analysis of its single-tryptophan variant W17A. The increase in the radius of gyration was associated with a single equilibrium unfolding transition in the case of two different denaturants, urea and guanidine hydrochloride (GuHCl). However, the compactness of urea- and GuHCl-unfolded molecules was comparable only at high denaturant concentrations and deviated under less denaturing conditions. Our results identified the role of conformational flexibility in IL-1ra aggregation and shed light on the nature of structural transitions within the folded ensembles of other beta-trefoil proteins, such as IL-1beta and hFGF-1.
Department of Analytical and Formulation Sciences, Amgen Inc., Seattle, Washington 98119, USA. rlatypov@amgen.com
Ramil F
RF
Latypov
Dingjiang
D
Liu
Jaby
J
Jacob
Timothy S
TS
Harvey
Pavel V
PV
Bondarenko
Gerd R
GR
Kleemann
David N
DN
Brems
Andrei A
AA
Raibekas
D002942:Circular Dichroism; D019286:Endopeptidase K / Q000737:chemistry; D005454:Fluorescence Polarization; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry; D014965:X-Rays
D053590:Interleukin 1 Receptor Antagonist Protein; D011994:Recombinant Proteins; D014508:Urea; D019286:Endopeptidase K; D019791:Guanidine