NMR studies of the backbone flexibility and structure of human growth hormone: a comparison of high and low pH conformations.
(15)N NMR relaxation parameters and amide (1)H/(2)H-exchange rates have been used to characterize the structural flexibility of human growth hormone (rhGH) at neutral and acidic pH. Our results show that the rigidity of the molecule is strongly affected by the solution conditions. At pH 7.0 the backbone dynamics parameters of rhGH are uniform along the polypeptide chain and their values are similar to those of other folded proteins. In contrast, at pH 2.7 the overall backbone flexibility increases substantially compared to neutral pH and the average order parameter approaches the lower limit expected for a folded protein. However, a significant variation of the backbone dynamics through the molecule indicates that under acidic conditions the mobility of the residues becomes more dependent on their location within the secondary structure units. In particular, the order parameters of certain loop regions decrease dramatically and become comparable to those found in unfolded proteins. Furthermore, the HN-exchange rates at low pH reveal that the residues most protected from exchange are clustered at one end of the helical bundle, forming a stable nucleus. We suggest that this nucleus maintains the overall fold of the protein under destabilizing conditions. We therefore conclude that the acid state of rhGH consists of a structurally conserved, but dynamically more flexible helical core surrounded by an aura of highly mobile, unstructured loops. However, in spite of its prominent flexibility the acid state of rhGH cannot be considered a "molten globule" state because of its high stability. It appears from our work that under certain conditions, a protein can tolerate a considerable increase in flexibility of its backbone, along with an increased penetration of water into its core, while still maintaining a stable folded conformation.
Kasimova
Marina R
MR
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.
Kristensen
Søren M
SM
Howe
Peter W A
PW
Christensen
Thorkild
T
Matthiesen
Finn
F
Petersen
Jørgen
J
Sørensen
Hans H
HH
Led
Jens J
JJ
D003903:Deuterium; D004355:Drug Stability; D013006:Growth Hormone / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008027:Light; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011522:Protons; D012542:Scattering, Radiation; D012996:Solutions; D013816:Thermodynamics
D011522:Protons; D012996:Solutions; D013006:Growth Hormone; D003903:Deuterium