THE DEPENDENCE OF CHEMICAL-EXCHANGE ON BOUNDARY SELECTION IN A FIBRONECTIN TYPE-III DOMAIN FROM HUMAN TENASCIN

The third fibronectin type III domain from human tenascin adopts a com pact beta-sandwich fold. Its boundaries were originally selected to en code a 90-residue domain (TNfn3(1-90)). We conclude that the dynamic p roperties of TNfn3 are more accurately represented when the C terminus is extended by the two naturally succeeding residues. Longitudinal (R -1) and transverse (R-2) N-15 relaxation rates, and {H-1-N-15} NOE enh ancements at pH 4.9 and 300 K are presented for TNfn3(1-90) and TNfn3( 1-92), the extended form, at two field strengths (11.74 and 14.10 T). Nearly identical results confirm their similar motional properties ove r a broad range of timescales. However, a number of residues near the C terminus in TNfn3(1-90) exhibit elevated transverse relaxation rates and broadened signals in H-1-N-15 HSQC spectra. Explicit rates of che mical exchange for five residues in TNfn3(1-90) were determined by mea suring transverse relaxation rates in a series of CPMG experiments wit h spin-echo refocusing delays increasing from 311 to 1436 mu s: Calcul ated exchange rates average 1000(+/-311) s(-1), with individual uncert ainties near 20%. Homonuclear TOCSY experiments collected between pH 4 and 7 reveal the coincident titration of two acidic clusters in TNfn3 (1-90) at pH 5.64(+/-0.47). The repulsive electrostatic interaction of the C-terminal carboxylate with one of these clusters may promote che mical exchange in the shorter domain. Additionally, NOE and chemical s hift data suggest hydrogen bond formation between the added residues a nd adjacent loops. The data, affirm the importance of judiciously sele cting domain boundaries prior to the characterization of molecular pro perties. (C) 1998 Academic Press.