CONFORMATIONAL DYNAMICS OF A MOBILE LOOP IN THE NAD(H)-BINDING SUBUNIT OF PROTON-TRANSLOCATING TRANSHYDROGENASES FROM RHODOSPIRILLUM-RUBRUMAND ESCHERICHIA-COLI

Transhydrogenase catalyses the reversible transfer of reducing equival ents between NAD(H) and NADP(H) to the translocation of protons across a membrane. Uniquely in Rhodospirillum rubrum, the NAD(H)-binding sub unit (called Th-s) exists as a separate subunit which can be reversibl y dissociated from the membrane-located subunits. We have expressed th e gene for R. rubrum Th-s in Escherichia coli to yield large quantitie s of protein. Low concentrations of either trypsin or endoproteinase L ys-C lead to cleavage of purified Th-s specifically at Lys227-Thr228 a nd Lys237-Glu238. Observations on the one-dimensional H-1-NMR spectra of Th-s before and after proteolysis indicate that the segment which s traddles the cleavage sites forms a mobile loop protruding from the su rface of the protein. Alanine dehydrogenase, which is very similar in sequence to the NAD(H)-binding subunit of transhydrogenase, lacks this segment. Limited proteolytic cleavage has little effect on some of th e structural characteristics of Th-s (its dimeric nature, its ability to bind to the membrane-located subunits of transhydrogenase, and the short-wavelength fluorescence emission of a unique Trp residue) but do es decrease the NADH-binding affinity, and does lower the catalytic ac tivity of the reconstituted complex. The presence of NADH protects aga inst trypsin or Lys-C cleavage, and leads to broadening, and in some c ases, shifting, of NMR spectral signals associated with amino acid res idues in the surface loop. This indicates that the loop becomes less m obile after nucleotide binding. Observation by NMR during a titration of Th-s with NAD(+) provides evidence of a two-step nucleotide binding reaction. By introducing an appropriate stop codon into the gene codi ng for the polypeptide of E. coli transhydrogenase cloned into an expr ession vector, we have prepared the NAD(H)-binding domain equivalent t o Th-s. The E. coli protein is sensitive to proteolysis by either tryp sin or Lys-C in the mobile loop. Judging by the effect of NADH on its NMR spectrum and on the fluorescence of its Trp residues, the protein is capable of binding the nucleotide though it is unable to dock with the membrane-located subunits of transhydrogenase from R. rubrum.