Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry.
An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used to extend previous hydrogen exchange studies on the position and energetic role of regulatory structure changes in hemoglobin. The results match earlier highly accurate but much more limited tritium exchange results, extend the analysis to the entire sequence of both hemoglobin subunits, and identify some energetically important changes. Allosterically sensitive amide hydrogens located at near amino acid resolution help to confirm the reality of local unfolding reactions and their use to evaluate resolved structure changes in terms of allosteric free energy.
Englander
Joan J
JJ
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA.
Del Mar
Charyl
C
Li
Will
W
Englander
S Walter
SW
Kim
Jack S
JS
Stranz
David D
DD
Hamuro
Yoshitomo
Y
Woods
Virgil L
VL
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003903:Deuterium / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D066298:In Vitro Techniques; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D021122:Protein Subunits; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
D006454:Hemoglobins; D010446:Peptide Fragments; D021122:Protein Subunits; D011506:Proteins; D006859:Hydrogen; D003903:Deuterium