Native-state hydrogen-exchange studies of a fragment complex can provide structural information about the isolated fragments.
Ordered protein complexes are often formed from partially ordered fragments that are difficult to structurally characterize by conventional NMR and crystallographic techniques. We show that concentration-dependent hydrogen exchange studies of a fragment complex can provide structural information about the solution structures of the isolated fragments. This general methodology can be applied to any bimolecular or multimeric system. The experimental system used here consists of Ribonuclease S, a complex of two fragments of Ribonuclease A. Ribonuclease S and Ribonuclease A have identical three-dimensional structures but exhibit significant differences in their dynamics and stability. We show that the apparent large dynamic differences between Ribonuclease A and Ribonuclease S are caused by small amounts of free fragments in equilibrium with the folded complex, and that amide exchange rates in Ribonuclease S can be used to determine corresponding rates in the isolated fragments. The studies suggest that folded RNase A and the RNase S complex exhibit very similar dynamic behavior. Thus cleavage of a protein chain at a single site need not be accompanied by a large increase in flexibility of the complex relative to that of the uncleaved protein.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560 012, India.
G
G
Chakshusmathi
G S
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Ratnaparkhi
P K
PK
Madhu
R
R
Varadarajan
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D006859:Hydrogen; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry*; D013381:Subtilisins / Q000737:chemistry
D010446:Peptide Fragments; D011522:Protons; D006859:Hydrogen; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; C022542:ribonuclease S; D013381:Subtilisins