Untitled

Abstract

Two triple resonance experiments, HNN and HN(C)N, are presented which correlate HN and 15N resonances sequentially along the polypeptide chain of a doubly (13C, 15N) labeled protein. These incorporate several improvements over the previously published sequences for a similar purpose and have several novel features. The spectral characteristics enable direct identification of certain triplets of residues, which provide many starting points for the sequential assignment procedure. The experiments are sensitive and their utility has been demonstrated with a 22 kDa protein under unfolding conditions where most of the standard triple resonance experiments such as HNCA, CBCANH etc. have limited success because of poor amide, Calpha and Cbeta chemical shift dispersions.

Folding Regulates Autoprocessing of HIV-1 Protease Precursor

“…The most significant feature of these experiments is the observation of different patterns of positive and negative peaks in the (F 1 , F 3 ) planes depending on the residue types at iϪ1, i, and iϩ1 positions. These have been discussed in detail earlier (39,40); suffice it to say here that glycines and prolines play important roles in this regard, the former because of the absence of the C ␤ , and the latter because of the absence of the amide proton. Triplets containing these residues produce very characteristic patterns in the (F 1 , F 3 ) planes, which can be termed as fixed points.…”

“…Uniformly 13 C]glucose. Protein was purified as described previously (35). MALDI analysis of the protein showed peaks at the expected molecular mass (17.3 kDa).…”

“…The next 99 residues, that is, 58 -156 actually constitute the PR portion. Hence residues 59 -62, 152-156 form the dimerization domain, 82-84 form the active site, 138 -140 form the substrate binding cleft, 100 -106 form the hinge region, and 109 -112 constitute the mobile flaps in the PR (35). The final five residues (157-161) having the sequence GGSSG, constitute an extension to the PR at the C terminus.…”

Sites of Tau Important for Aggregation Populate β-Structure and Bind to Microtubules and Polyanions

“…Therefore, an unambiguous assignment was not possible just relying on the conventional three-dimensional spectra HNCO, HN(CA)CO, HNCACB and CBCA(CO)NH (19). A high-resolution (HA)CANNH (20) together with three-dimensional HNN and HN(C)N experiments were required (21). With these NMR spectra the backbone resonance assignment of K18 and K19 could be achieved in an iterative fashion based on automatic assignment using the program MARS (22) and manual assignment mainly relying on the HNN experiment.…”

Structural Dynamics and Multiregion Interactions in Dynein-Dynactin Recognition

“…Excluded residues correspond to overlapped peaks or to peaks too weak for reliable measurement. The majority of residues included in the analysis exhibit 3 J-coupling values in the 6 -8 Hz range, indicative of disordered conformations; however, there also exist contiguous stretches of residues (3-9, 11-23, and [27][28][29][30][31][32][33][34][35][36][37] NOEs between residues 32 and 34 (Fig. 3).…”