Pockets of short-range transient order and restricted topological heterogeneity in the guanidine-denatured state ensemble of GED of dynamin.
The nature and variety in the denatured state of a protein, a non-native state under a given set of conditions, has been a subject of intense debate. Here, using multidimensional NMR, we have characterized the 6 M Gdn-HCl-denatured state of GED, the assembly domain of dynamin. Even under such strongly denaturing conditions, we detected the presence of conformations in slow exchange on the NMR chemical shift time scale. Although the GED oligomer as well as the SDS-denatured monomeric GED were seen to be predominantly helical [Chugh et al. (2006) FEBS J. 273, 388-397], the 6 M Gdn-HCl-denatured GED has largely beta-structural preferences. However, against such a background, we could detect the presence of a population with a short helical stretch (Arg42-Ile47) in the ensemble. The 1H-1H NOEs suggested presence of pockets of transient short-range order along the chain. Put together these segments may lead to a rather small number of interconverting topologically distinguishable ensembles. Spectral density analysis of 15N relaxation rates and {1H}-15N NOE, measured at 600 and 800 MHz, and comparison of J(0) with hydrophobic patches calculated using AABUF approach, indicated presence of four domains of slow motions. These coincided to a large extent with those showing significant Rex. Additionally, a proline residue in the connection between two of these domains seems to cause a fast hinge motion. These observations help enhance our understanding of protein denatured states, and of folding concepts, in general.
Chugh
Jeetender
J
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005, India.
Sharma
Shilpy
S
Hosur
Ramakrishna V
RV
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D003903:Deuterium; D034284:Dynamin I / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D019791:Guanidine / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010957:Plasmids; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
D018076:DNA, Complementary; D009587:Nitrogen Isotopes; D011392:Proline; D003903:Deuterium; D034284:Dynamin I; D019791:Guanidine