The herpes simplex virus triplex protein, VP23, exists as a molten globule.

Two proteins, VP19C (50,260 Da) and VP23 (34,268 Da), make up the triplexes which connect adjacent hexons and pentons in the herpes simplex virus type 1 capsid. VP23 was expressed in Escherichia coli and purified to homogeneity by Ni-agarose affinity chromatography. In vitro capsid assembly experiments demonstrated that the purified protein was functionally active. Its physical status was examined by differential scanning calorimetry, ultracentrifugation, size exclusion chromatography, circular dichroism, fluorescence spectroscopy, and 8-anilino-1-naphthalene sulfonate binding studies. These studies established that the bacterially expressed VP23 exhibits properties consistent with its being in a partially folded, molten globule state. We propose that the molten globule represents a functionally relevant intermediate which is necessary to allow VP23 to undergo interaction with VP19C in the process of capsid assembly.

Stopped-flow analysis of the refolding of hen egg white riboflavin binding protein in its native and dephosphorylated forms.

In earlier work (D.A. McClelland, S.H. McLaughlin, R.B. Freedman, and N.C. Price, Biochem. J. 311, 133-137 (1995)), we had shown that during the refolding of hen egg white riboflavin binding protein (RfBP) after denaturation in 6 M guanidinium chloride, most of the native properties of the protein are regained within 10-15 s of the dilution of the denaturing agent. We have now employed stopped-flow measurements of CD, protein fluorescence and regain of riboflavin binding ability to examine the rapid phases of the refolding process. Essentially, all of the native secondary structure as judged by the CD signal at 230 nm was regained within the dead-time of the instrument in CD mode (approximately 8 ms). 80% of the native protein fluorescence was regained within the dead-time of the instrument in fluorescence mode (1.7 ms). A further 10% was regained with a half time of 30 ms in the case of the apo-protein, though the half time was approximately doubled in the presence of riboflavin. This second phase corresponded with the regain of riboflavin binding ability. Two slow phases, with half-times of 46 s and 1 h involved the regain of the final 10% of fluorescence signal. Binding of the fluorescent probe, 1-anilino-8-naphthalenesulphonate (ANS) preceded the formation of the riboflavin binding site. Dephosphorylation of RfBP by treatment with acid phosphatase did not affect the binding of riboflavin, nor did it alter the kinetics of the refolding process. This is consistent with the proposal that in vivo phosphorylation occurs on a surface-exposed portion of the protein after the major portion of the folding process is complete.

The refolding of hen egg white riboflavin-binding protein: effect of protein disulphide isomerase on the reoxidation of the reduced protein.

Hen egg white riboflavin-binding protein (RfBP) contains nine disulphide bonds. Provided these remain intact, the refolding of RfBP after incubation in 6 M guanidinium chloride is highly efficient with at least 95% of the binding activity regained within 3 min. Kinetic studies indicate that this regain consists of at least two phases. When the disulphide bonds of RfBP are reduced, reoxidation using a mixture of oxidized and reduced glutathione leads to less than 5% recovery of activity. However, if protein disulphide isomerase (PDI; EC 5.3.4.1) is present during the reoxidation nearly 50% activity can be regained, suggesting that PDI may play an important role in the maturation of RfBP in vivo.

The total N-acetyl neuraminic acid content of human normal and lymphatic leukaemic lymphocytes.

The total N-acetyl neuraminic acid (NANA) content of lymphocytes, erythrocytes and plasma obtained from normal and lymphatic leukaemic patients was measured by the Warren thiobarbiturate technique. Lymphocytes from patients with chronic lymphatic leukaemia and lymphoblasts from patients with acute leukaemia had decreased levels.