High-resolution probing of local conformational changes in proteins by the use of multiple labeling: unfolding and self-assembly of human carbonic anhydrase II monitored by spin, fluorescent, and chemical reactivity probes.
Two different spin labels, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (IPSL) and (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL), and two different fluorescent labels 5-((((2-iodoacetyl)amino)-ethyl)amino)naphtalene-1-sulfonic acid (IAEDANS) and 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN), were attached to the introduced C79 in human carbonic anhydrase (HCA II) to probe local structural changes upon unfolding and aggregation. HCA II unfolds in a multi-step manner with an intermediate state populated between the native and unfolded states. The spin label IPSL and the fluorescent label IAEDANS reported on a substantial change in mobility and polarity at both unfolding transitions at a distance of 7.4-11.2 A from the backbone of position 79. The shorter and less flexible labels BADAN and MTSSL revealed less pronounced spectroscopic changes in the native-to-intermediate transition, 6.6-9.0 A from the backbone. At intermediate guanidine (Gu)-HCl concentrations the occurrence of soluble but irreversibly aggregated oligomeric protein was identified from refolding experiments. At approximately 1 M Gu-HCl the aggregation was found to be essentially complete. The size and structure of the aggregates could be varied by changing the protein concentration. EPR measurements and line-shape simulations together with fluorescence lifetime and anisotropy measurements provided a picture of the self-assembled protein as a disordered protein structure with a representation of both compact as well as dynamic and polar environments at the site of the molecular labels. This suggests that a partially folded intermediate of HCA II self-assembles by both local unfolding and intermolecular docking of the intermediates vicinal to position 79. The aggregates were determined to be 40-90 A in diameter depending on the experimental conditions and spectroscopic technique used.
Hammarström
P
P
Department of Chemistry, Linköping University, SE-581 83 Linköping, Sweden.
Owenius
R
R
Mårtensson
L G
LG
Carlsson
U
U
Lindgren
M
M
D019943:Amino Acid Substitution / Q000235:genetics; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003198:Computer Simulation; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004058:Diffusion; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D015335:Molecular Probes / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D012399:Rotation; D013113:Spin Labels; D055672:Static Electricity; D013329:Structure-Activity Relationship
D005456:Fluorescent Dyes; D015335:Molecular Probes; D013113:Spin Labels; D002256:Carbonic Anhydrases; D003545:Cysteine