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Human TFIIB, an essential factor in transcription of protein-coding genes by RNA polymerase II, consists of an amino-terminal zinc binding domain (TFIIBn) connected by a linker of about 60 residues to a carboxy-terminal core domain (TFIIBc). The TFIIB core domain has two internally repeated motifs, each comprising five alpha-helices arranged as in the cyclin box. Compared to the crystal structure of TFIIBc in complex with TBP and a TATA-containing oligonucleotide, the NMR-derived solution structure of free TFIIBc is more compact, with a different repeat-repeat orientation and a significantly shorter first helix in the second repeat. Analysis of backbone 15N relaxation parameters indicates the presence of relatively large amplitude, nanosecond time-scale motions in the TFIIBc interrepeat linker and structural fluctuations throughout the backbone. Interaction of TFIIBc with the acidic activation domain of VP16 or with TFIIBn induces 1H-15N chemical shift and line width changes concentrated in the first repeat, interrepeat linker and the first helix of the second repeat. These results suggest that TFIIB is somewhat pliable and that the conformation of the C-terminal core domain can be modulated by interaction with the N-terminal zinc binding domain. Furthermore, binding of the VP16 activation domain may promote TFIIBc conformations primed for binding to a TBP-DNA complex.

The constant fragment of the immunoglobulin light chain (type lambda) has two trytophyl residues at positions 150 and 187. Trp-150 is buried in the interior, and Trp-187 lies on the surface of the molecule. The hydrogen-deuterium exchange kinetics of the indole NH proton Trp-150 were studied at various pH values at 25/sup 0/C by /sup 1/H nuclear magnetic resonance. Exchange rates were approximately first order in hydroxyl ion dependence above pH 8, were relatively independent of pH between pH 7 and 8, and decreased below pH 7. On the assumption that the exchange above pH 8 proceeds through local fluctuations of the protein molecule, the exchange rates between pH 7 and 8 through global unfolding were estimated. The exchange rate constant within this pH range at 25/sup 0/C thus estimated was consistent with that of the global unfolding of the constant fragment under the same conditions as those reported previously. The activation energy for the exchange process at pH 7.8 was the same as that for the unfolding process by 2 M guanidine hydrochloride. The exchange rates of backbone NH protons were almost the same as that of the indole NH proton of Trp-150 at pH 7.l. These observations alsomore » indicated that the exchange between pH 7 and 8 occurs through global unfolding of the protein molecule and is rate-limited by the unfolding. At around pH 9, on the other hand, the activation energy for the exchange process of the indole NH proton of Trp-150 was smaller than that for the unfolding process, and the exchange rates differed according to the different signals of backbone NH protons. These findings together with the pH dependence of the rate constant indicated that exchange due to local fluctuations is predominant above pH 8.« less

The results of photo-CIDNP measurements of Bence Jones proteins, immunoglobulins, and their proteolytic fragments are reported. The CIDNP spectra of constant region CL fragments, which were derived from lambda-type Bence Jones proteins with three different isotypes, gave polarizations originating from two Tyr and one Trp residues. From comparisons of the results obtained by using lambda-type Bence Jones proteins that possess the known amino acid sequences with those for the CL fragments, it was concluded that the CIDNP signals observed in the case of Bence Jones proteins are all due to the hypervariable region. On the basis of the results obtained by using IgG1, IgG2, IgG3, and IgG4 myeloma proteins along with their Fab, Fc, and pFc' fragments, it was concluded that CIDNP signals for the Fab region all originate from the variable region. The Fc region gives two Tyr polarizations, one of which has been assigned to Tyr-296. We have also shown that the second Tyr signal is due to Tyr-373. Interaction of Fc and staphylococcal protein A has also been examined by the CIDNP technique.