The dependence of chemical exchange on boundary selection in a fibronectin type III domain from human tenascin

  • Meekhof A
  • Hamill S
  • Arcus V
 et al. 
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Abstract

The third fibronectin type III domain from human tenascin adopts a compact β-sandwich fold. Its boundaries were originally selected to encode a 90-residue domain (TNfn31-90). We conclude that the dynamic properties of TNfn3 are more accurately represented when the C terminus is extended by the two naturally succeeding residues. Longitudinal (R1) and transverse (R2)15N relaxation rates, and {1H-15N} NOE enhancements at pH 4.9 and 300 K are presented for TNfn31-90and TNfn31-92, the extended form, at two field strengths (11.74 and 14.10 T). Nearly identical results confirm their similar motional properties over a broad range of timescales. However, a number of residues near the C terminus in TNfn31-90exhibit elevated transverse relaxation rates and broadened signals in1H-15N HSQC spectra. Explicit rates of chemical exchange for five residues in TNfn31-90were determined by measuring transverse relaxation rates in a series of CPMG experiments with spin-echo refocusing delays increasing from 311 to 1436 μs. Calculated exchange rates average 1000 (±311) s-1, with individual uncertainties near 20%. Homonuclear TOCSY experiments collected between pH 4 and 7 reveal the coincident titration of two acidic clusters in TNfn31-90at pH 5.64 (±0.47). The repulsive electrostatic interaction of the C-terminal carboxylate with one of these clusters may promote chemical exchange in the shorter domain. Additionally, NOE and chemical shift data suggest hydrogen bond formation between the added residues and adjacent loops. The data affirm the importance of judiciously selecting domain boundaries prior to the characterization of molecular properties.

Author-supplied keywords

  • Backbone dynamics
  • Boundary selection
  • Domain
  • Fibronectin type III
  • NMR

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