Mapping the ATP binding site in the plasma membrane H(+)-ATPase from Kluyveromyces lactis.

The plasma membrane H(+)-ATPase from Kluyveromyces lactis contains 14 tryptophan residues. Binding a nucleotide or unfolding with Gnd-HCl quenched intrinsic fluorescence by ≈60% suggesting that in the H(+)-ATPase-Nucleotide complex there is solvent-mediated collisional quenching of W505 fluorescence. N-bromosuccinimide (NBS) treatment of H(+)-ATPase modified a single W residue in both native and Gnd-HCl-unfolded H(+)-ATPase. Denaturing the H(+)-ATPase with 1% SDS led to expose six tryptophan residues while requiring 17 NBS/H(+)-ATPase. The remaining eight tryptophan residues kept buried indicating a highly stable TM domain. Acrylamide generated static quenching of fluorescence; partial in the native enzyme (V = 0.43 M(-1)) and complete in the Gnd-HCl-unfolded H(+)-ATPase (V = 0.81 M(-1)). Collisional quenching (K sv) increased from 3.12 to 7.45 M(-1) upon H(+)-ATPase unfolding. W505 fluorescence titration with NBS yielded a molar ratio of 6 NBS/H(+)-ATPase and quenched ≈ 60% fluorescence. In the recombinant N-domain, the distance between W505 and MantATP was estimated to be 21 Å by FRET. The amino acid residues involved in nucleotide binding were identified by N-domain molecular modelling and docking with ATP. In the N-domain/ATP complex model, the distance between W505 and ATP was 20.5 Å. ATP binding leads to a conformational change in the N-domain of H(+)-ATPase that exposes W505 to the environment.

Pea lectin unfolding reveals a unique molten globule fragment chain.

Pea lectin (PSL) is a dimeric protein in which each subunit comprises two intertwined, post-translationally processed polypeptide chains--a long ß-fragment and a short α-fragment. Using guanidine hydrochloride-induced denaturation, we have investigated and characterized the species obtained in the unfolding equilibrium of PSL by steady-state and time-resolved fluorescence, phosphorescence, and selective chemical modification. During unfolding, the fragment chains become separated, and the unfolding pattern reveals a ß-fragment as intermediate that has the molten globule characteristics. As examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, the fragment intermediate shows ~20 fold increase in ANS fluorescence, and a large increase in ANS lifetime (12.8 ns). The tryptophan environment of the molten globule ß-fragment has been probed by selective modification with N-bromosuccinimide (NBS), which shows that two tryptophans, possibly Trp 53 and Trp 152 are oxidized while the other Trp 128 remains resistant to oxidation. The different types of tryptophan environment for the intermediate are supported by phosphorescence studies at 77 K, which gives a (0,0) band at 410 nm. These results seem to indicate that the larger fragment chain of PSL can independently behave as a monomeric or single domain protein that undergoes unfolding through intermediate state(s), and may provide important insight into the folding problem of oligomeric proteins in general and lectins in particular.

A mannose/glucose-specific lectin from Chinese evergreen chinkapin (Castanopsis chinensis).

A mannose/glucose-specific lectin has been purified from Chinese evergreen chinkapin (Castanopsis chinensis) seeds, one of the most popular foods in East Asia. This lectin, designated as CCL, exhibited hemagglutinating activity in mouse and rabbit erythrocytes. It displayed a single band with a molecular mass of 29 kDa in SDS-PAGE and a 120-kDa peak in gel-filtration on Superdex-200. Its hemagglutinating activity was stable in the pH range 6-12 and at temperatures below 60 degrees C. The N-terminal amino acid sequence of CCL differed from those of other lectins in the same family. CCL inhibited the proliferation of HepG2 cells and adult emergence in fruitflies. CCL exhibited mitogenic activity toward mouse splenocytes, and induced nitric oxide production from mouse peritoneal macrophages but was devoid of inhibitory activity toward mycelial growth and HIV-1 reverse transcriptase.

Cysteine-containing histone H1-like (PL-I) proteins of sperm.

We have determined the presence of cysteine in the protein PL-I from the sperm of the surf clam Spisula solidissima. The existence of cysteine in this histone H1-related protein is responsible for its previously described aggregation behavior. The location of this residue, within the trypsin-resistant domain of the protein, has been established. We have also shown that cysteine is ubiquitously present in the PL-I proteins from the sperm of other bivalve mollusks but is absent from other PL of smaller molecular mass (PL-II, PL-III, PL-IV). We have also found cysteine to be present in the PL-I from a tunicate (Chelysoma productum) but absent in a PL-I from a fish (Mullus barbatus). The possible significance of the unusual occurrence of cysteine in these histone-H1-related proteins is discussed.

The role of tryptophanyl residues in electron transfer from NADPH--adrenodoxin reductase to adrenodoxin.

Chemical modification of tryptophanyl residues of NADPH - adrenodoxin reductase by N - bromosuccinimide and trichloroethanol prevents the interaction of the enzyme with adrenodoxin. The modification does not touch other amino acid residues besides tryptophan (tyrosine, lysine and cysteine) or disturb the structure of protein. The presence of adrenodoxin suppresses the modification. The data obtained indicate the participation of adrenodoxin reductase tryptophan residues in the interaction with adrenodoxin.