Independently melting modules and highly structured intermodular junctions within complement receptor type 1.
A segment of complement receptor type 1 (CR1) corresponding to modules 15-17 was overexpressed as a functionally active recombinant protein with N-glycosylation sites ablated by mutagenesis (referred to as CR1 approximately 15-17(-)). A protein consisting of modules 15 and 16 and another corresponding to module 16 were also overexpressed. Comparison of heteronuclear nuclear magnetic resonance (NMR) spectra for the single, double, and triple module fragments indicated that module 16 makes more extensive contacts with module 15 than with module 17. A combination of NMR, differential scanning calorimetry, circular dichroism, and tryptophan-derived fluorescence indicated a complex unfolding pathway for CR1 approximately 15-17(-). As temperature or denaturant concentration was increased, the 16-17 junction appeared to melt first, followed by the 15-16 junction, and module 17 itself; finally, modules 15 and 16 became denatured. Modules 15 and 16 adopted an intermediate state prior to total denaturation. These results are compared with a previously published study [Clark, N. S., Dodd, I, Mossakowska, D. E., Smith, R. A. G., and Gore, M. G. (1996) Protein Eng. 9, 877-884] on a fragment consisting of the N-terminal three CR1 modules which appeared to melt as a single unit.
Kirkitadze
M D
MD
The Edinburgh Centre for Protein Technology, Scotland.
Krych
M
M
Uhrin
D
D
Dryden
D T
DT
Smith
B O
BO
Cooper
A
A
Wang
X
X
Hauhart
R
R
Atkinson
J P
JP
Barlow
P N
PN
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D017463:Receptors, Complement 3b / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D010446:Peptide Fragments; D017463:Receptors, Complement 3b; D011994:Recombinant Proteins; D012996:Solutions