title: Characterizing the use of perdeuteration in NMR studies of large proteins: 13C, 15N and 1H assignments of human carbonic anhydrase II.
abstract: Perdeuteration of all non-exchangeable proton sites can significantly increase the size of proteins and protein complexes for which NMR resonance assignments and structural studies are possible. Backbone 1H, 15N, 13CO, 13C alpha and 13C beta chemical shifts and aliphatic side-chain 13C and 1H(N)/15N chemical shifts for human carbonic anhydrase II (HCA II), a 259 residue 29 kDa metalloenzyme, have been determined using a strategy based on 2D, 3D and 4D heteronuclear NMR experiments, and on perdeuterated 13C/15N-labeled protein. To date, HCA II is one of the largest monomeric proteins studied in detail by high-resolution NMR. Of the backbone resonances, 85% have been assigned using fully protonated 15N and 3C/15N-labeled protein in conjunction with established procedures based on now standard 2D and 3D NMR experiments. HCA II has been perdeuterated both to complete the backbone resonance assignment and to assign the aliphatic side-chain 13C and 1H(N)/15N resonances. The incorporation of 2H into HCA II dramatically decreases the rate of 13C and 1H(N)T2 relaxation. This, in turn, increases the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments. Many otherwise marginal heteronuclear 3D and 4D correlation experiments, which are important to the assignment strategy detailed herein, can now be executed successfully on HCA II. Further analysis suggests that, from the perspective of sensitivity, perdeuteration should allow other proteins with rotational correlation times significantly longer than HCA II (tau c = 11.4 ns) to be studied successfully with these experiments. Two different protocols have been used to characterize the secondary structure of HCA II from backbone chemical-shift data. Secondary structural elements determined in this manner compare favorably with those elements determined from a consensus analysis of the HCA II crystal structure. Finally, having outlined a general strategy for assigning backbone and side-chain resonances in a perdeuterated large protein, we propose a strategy whereby this information can be used to glean more detailed structural information from the partially or fully protonated protein equivalent.
authors.affiliation: Duke University NMR Center, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Venters
authors.forename: B T
authors.initials: BT
authors.lastname: Farmer
authors.forename: C A
authors.initials: CA
authors.lastname: Fierke
authors.forename: L D
authors.initials: LD
authors.lastname: Spicer
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D002247:Carbon Isotopes; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics; D003903:Deuterium; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006859:Hydrogen; D015394:Molecular Structure; D009587:Nitrogen Isotopes; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D000596:Amino Acids; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D011522:Protons; D011994:Recombinant Proteins; D006859:Hydrogen; D003903:Deuterium; D002256:Carbonic Anhydrases