Biophysical analysis of natural variants of the multimerization region of Epstein-Barr virus lytic-switch protein BZLF1.

BZLF1 plays a key role in the induction of Epstein-Barr virus (EBV) replication. On the basis of limited sequence homology and mutagenesis experiments, BZLF1 has been described as a member of the bZip family of transcription factors, but this prospect has not been rigorously tested to date. Here, we present biophysical analysis of the multimerization domain of BZLF1, from three natural variants of EBV, and demonstrate for the first time that the region between amino acids 196 and 227 is sufficient to direct folding as a coiled-coil dimer in vitro.

Sticky-end assembly of a designed peptide fiber provides insight into protein fibrillogenesis.

Coiled-coil motifs provide simple systems for studying molecular self-assembly. We designed two 28-residue peptides to assemble into an extended coiled-coil fiber. Complementary interactions in the core and flanking ion-pairs were used to direct staggered heterodimers. These had "sticky-ends" to promote the formation of long fibers. For comparison, we also synthesized a permuted version of one peptide to associate with the other peptide and form canonical heterodimers with "blunt-ends" that could not associate longitudinally. The assembly of both pairs was monitored in solution using circular dichroism spectroscopy. In each case, mixing the peptides led to increased and concentration-dependent circular dichroism signals at 222 nm, consistent with the desired alpha-helical structures. For the designed fiber-producing peptide mixture, we also observed a linear dichroism effect during flow orientation, indicative of the presence of long fibrous structures. X-ray fiber diffraction of partially aligned samples gave patterns indicative of coiled-coil structure. Furthermore, we used electron microscopy to visualize fiber formation directly. Interestingly, the fibers observed were at least several hundred micrometers long and 20 times thicker than expected for the dimeric coiled-coil design. This additional thickness implied lateral association of the designed structures. We propose that complementary features present in repeating structures of the type we describe promote lateral assembly, and that a similar mechanism may underlie fibrillogenesis in certain natural systems.

Structural characterization of a methionine-rich, emulsifying protein from sunflower seed.

The 2 S seed storage protein, sunflower albumin 8, contains an unusually high proportion of hydrophobic residues including 16 methionines in a mature protein of 103 amino acids. A structural model, based on the known structure of a related protein, has been constructed as a four-helix bundle cross-linked by four disulphide bonds. This model structure is consistent with data from circular dichroism and nuclear magnetic resonance experiments. Analysis of the model's surface shows the presence of a large hydrophobic face that may be responsible for the highly stable emulsions this protein is known to form with oil/water mixtures.

Direct kinetic evidence for folding via a highly compact, misfolded state.

The 2 S seed storage protein, sunflower albumin 8 (SFA-8), contains an unusually high proportion of hydrophobic residues including 16 methionines (some of which may form a surface hydrophobic patch) in a disulfide cross-linked, alpha-helical structure. Circular dichroism and fluorescence spectroscopy show that SFA-8 is highly stable to denaturation by heating or chaotropic agents, the latter resulting in a reversible two-state unfolding transition. The small m(U) (-4.7 M(-1) at 10 degrees C) and DeltaC(p) (-0.95 kcal mol(-1) K(-1)) values indicate that relatively little nonpolar surface of the protein is exposed during unfolding. Commensurate with the unusual distribution of hydrophobic residues, stopped-flow fluorescence data show that the folding pathway of SFA-8 is highly atypical, in that the initial product of the rapid collapse phase of folding is a compact nonnative state (or collection of nonnative states) that must unfold before acquiring the native conformation. The inhibited folding reaction of SFA-8, in which the misfolded state (m(M) = -0.95 M(-1) at 10 degrees C) is more compact than the transition state for folding (m(T) = -2.5 M(-1) at 10 degrees C), provides direct kinetic evidence for the transient misfolding of a protein.

Unusual presentation of T-cell lymphoma.

A 56-year old woman developed multiple papules, plaques and erythema - multiforme (EM) like lesions on palms, soles, abdomen, back, axillary and gluteal folds of one month duration. She also had erythematous plaques, painful necrotising ulcers and oro-genital ulcerations. Skin biopsy was suggestive of plaque stage of cutaneous T-cell lymphoma.

Complete amino acid sequences of two trypsin inhibitors from buckwheat seed.

The major trypsin isoinhibitors from seed extracts of buckwheat (Fagopyrum esculentum Mönch) were purified by affinity chromatography, anion exchange chromatography, anion exchange HPLC and reversed-phase HPLC, and the complete amino acid sequences of two isoinhibitors, BTI-1 and BTI-2, were established by automated Edman degradation. Each isoinhibitor consists of a single polypeptide chain of 69 amino acids, including two Cys residues. The N-terminal sequence of a third isoform, BTI-3, was also determined. The buckwheat trypsin isoinhibitors exhibit clear sequence similarities with the potato chymotrypsin inhibitor I family of serine proteinase inhibitors.