Fractional 13C enrichment of isolated carbons using [1-13C]- or [2- 13C]-glucose facilitates the accurate measurement of dynamics at backbone Calpha and side-chain methyl positions in proteins.
A simple labeling approach is presented based on protein expression in [1-(13)C]- or [2-(13)C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C(alpha) sites, respectively. All of the methyl groups, with the exception of Thr and Ile(delta1) are produced with isolated (13)C spins (i.e., no (13)C-(13)C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-alpha sites are labeled without concomitant labeling at C(beta) positions for 17 of the common 20 amino acids and there are no cases for which (13)C(alpha)-(13)CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone (15)N studies. The utility of the labeling is established by recording (13)C R (1rho) and CPMG-based experiments on a number of different protein systems.
Lundström
Patrik
P
Department of Medical Genetics, The University of Toronto, Toronto, ON, Canada, M5S 1A8.
Teilum
Kaare
K
Carstensen
Tommy
T
Bezsonova
Irina
I
Wiesner
Silke
S
Hansen
D Flemming
DF
Religa
Tomasz L
TL
Akke
Mikael
M
Kay
Lewis E
LE
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000096:biosynthesis / Q000737:chemistry; D000818:Animals; D002244:Carbon / Q000032:analysis* / Q000737:chemistry*; D002247:Carbon Isotopes; D002417:Cattle; D005947:Glucose / Q000737:chemistry*; D006801:Humans; D008697:Methane / Q000031:analogs & derivatives* / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*
D000596:Amino Acids; D002247:Carbon Isotopes; D011506:Proteins; C051224:methyl radical; D002244:Carbon; D005947:Glucose; D008697:Methane