A quantitative description of in vitro assembly of human papillomavirus 16 virus-like particles.

The full-length human papillomavirus 16 major capsid protein L1 is expressed in Saccharomyces cerevisiae as virus-like particles (VLPs). However, yeast-expressed human papillomavirus 16 particles are irregular in shape and are prone to aggregate. When disassembled and reassembled, the resulting particles have improved stability and solubility. We have examined VLP dissociation and reassembly to define the important features of the assembly mechanism. We found that the VLPs rapidly disassemble at pH 8.2 and low ionic strength in the presence of low concentrations of reducing agents. The pH dependence of assembly kinetics and extent of assembly under reducing conditions were differentially sensitive to ionic strength. Assembly at pH 5.2 was very fast and led to heavily aggregated particles. This sort of kinetic trap is expected for overinitiated assembly. We observed that reassembly at pH 6.2, 7.2, and 8.2 yielded regular particles over a broad range of ionic strength. At these three pH values, assembly was quantitative at 1 M NaCl. At pH 7.2, much more than at pH 6.2 or pH 8.2, assembly decreased monotonically with ionic strength. The free energy of association ranged from -8 to -10 kcal/mol per pentamer. The effect of pH on assembly was further investigated by examining dissociation of reassembled particles. Though indistinguishable by negative stain electron microscopy, particles assembled at pH 7.2 disassembled slower than pH 5.2, 6.2, or 8.2 VLPs. We hypothesize that pH 7.2 assembly reactions lead to formation of particles with conformationally different interactions.

5-Dodecanoylaminofluorescein as a probe for the determination of critical micelle concentration of detergents using fluorescence anisotropy.

A method for determining the critical micelle concentration (CMC) of various detergents based on fluorescence polarization (anisotropy) of the lipophilic probe 5-dodecanoylaminofluorescein is presented. Nonionic, cationic, anionic, and steroid-based detergents can all be evaluated by this method and the determined CMC values of selected detergents agree well with those reported in the literature. In addition, we report the CMC of domiphen bromide, whose CMC value has not previously been described. In the case of ionic detergents, the method described is particularly sensitive at discerning changes in the CMC with increasing ionic strength of the medium and can discriminate detergent CMCs in 5 mM versus 25 mM buffering components. The described fluorescence polarization technique allows very low (submicromolar) concentrations of probe to be employed, thus minimizing the perturbation of micelle formation by 5-dodecanoylaminofluorescein insertion.

Dual roles of an E-helix residue, Glu167, in the transcriptional activator function of CooA.

CooA is a transcriptional activator that mediates CO-dependent expression of the genes responsible for CO oxidation in Rhodospirillum rubrum. In this study, we suggest in vitro and in vivo models explaining an unusual requirement of CooA for millimolar levels of divalent cations for high-affinity DNA binding. Several lines of evidence indicate that an E-helix residue, Glu167, plays a central role in this requirement by inhibiting sequence-specific DNA binding via charge repulsion in the absence of any divalent cation and that divalent cations relieve such repulsion in the process of DNA binding by CooA. Unexpectedly, the Glu167 residue is the optimal residue for in vivo transcriptional activity of CooA. We present a model in which the Glu167 from the downstream subunit of CooA helps the protein to interact with RNA polymerase, probably through an interaction between activating region 3 and sigma subunit. The study was further extended to a homologous protein, cyclic AMP receptor protein (CRP), which revealed similar, but not identical, roles of the residue in this protein as well. The results show a unique mechanism of CooA modulating its DNA binding and transcriptional activation in response to divalent cations among the CRP/FNR (fumarate and nitrate reductase activator protein) superfamily of regulators.

An automated homogeneous method for quantifying polysorbate using fluorescence polarization.

An automated fluorescence polarization (FP) assay has been developed for the quantitation of polysorbate in bioprocess samples. Using the lipophilic probe 5-dodecanoylaminofluorescein (DAF), polysorbate concentrations above the critical micelle concentration can be quantified by the FP increase that results when DAF inserts into the detergent micelles. The specificity, accuracy, and precision of this assay were defined for samples obtained from vaccine purification processes. Spike recoveries were 98-106% for purified products and 110-120% for crude process intermediates. The coefficients of variation for intra- and interassay precision were less than 9 and 14%, respectively. Because of the operational simplicity of the assay, all of the assay steps from sample preparation to data reduction were automated on a Tecan liquid-handling workstation. The combination of a rapid assay and an automated format makes this method well suited to the routine analysis of samples from trial purification processes which are carried out during the development of a vaccine or therapeutic protein. This method should be adaptable for the quantitation of other detergents into which DAF will insert.

Dynamics of carbon monoxide binding to CooA.

CooA is a dimeric CO-sensing heme protein from Rhodospirillum rubrum. The heme iron in reduced CooA is six-coordinate; the axial ligands are His-77 and Pro-2. CO displaces Pro-2 and induces a conformation change that allows CooA to bind DNA and activate transcription of coo genes. Equilibrium CO binding is cooperative, with a Hill coefficient of n = 1.4, P(50) = 2.2 microm, and estimated Adair constants K(1) = 0.16 and K(2) = 1.3 microm(-1). The rates of CO binding and release are both strongly biphasic, with roughly equal amplitudes for the fast and slow phases. The association rates show a hyperbolic dependence on [CO], consistent with Pro-2 dissociation being rate-limiting. The kinetic characteristics of the transiently formed five-coordinate heme are probed via flash photolysis. These observations are integrated into a kinetic model, in which CO binding to one subunit decreases the rate of Pro-2 rebinding in the second, leading to a net increase in affinity for the second CO. The CO adduct exists in slowly interconverting "open" and "closed" forms. This interconversion probably involves the large-scale motions required to bring the DNA-binding domains into proper orientation. The combination of low CO affinity, slow CO binding, and slow conformational transitions ensures that activation of CooA only occurs at high (micromolar) and sustained (> or =1 min) levels of CO. When micromolar levels do occur, positive cooperativity allows efficient activation over a narrow range of CO concentrations.

Repositioning about the dimer interface of the transcription regulator CooA: a major signal transduction pathway between the effector and DNA-binding domains.

Activation of the homodimeric transcriptional regulator CooA depends on the coupling of CO binding at an effector domain heme with the allosteric repositioning of the DNA-binding domain F-helix that promotes specific DNA interaction. By analogy to the homologous cAMP receptor protein (CRP), it has been proposed that effector binding elicits subunit reorientation about their coiled-coil C-helix interface, and that this effector domain reorientation stabilizes the active position of the DNA-binding domains. Here, we describe experiments in which effector-independent "CooA*" variants were selected following randomization of a six-residue portion of the C-helix dimerization domain. Subsequent activity analyses, both in vivo and in vitro, were consistent with a model wherein improved C-helix "leucine zipper" interactions modestly shifted the regulator population equilibrium towards the active conformation, although full activation remained CO-dependent. However, in addition to the improved leucine zipper, maximal CooA* activity required additional C-helix changes which in a WT background decreased normal CO-dependent DNA-binding 100-fold. This seemingly paradoxical combination suggested that maximal CooA* activity depended both on the improved coiled-coil interactions and the decoupling of the signal pathway within the effector domain. Both types of C-helix changes indicate that its repositioning is crucial for the allosteric shift in the inactive/active equilibrium of the DNA-binding domain.

Analysis of the L116K variant of CooA, the heme-containing CO sensor, suggests the presence of an unusual heme ligand resulting in novel activity.

CooA is the CO-sensing transcriptional activator from Rhodospirillum rubrum, in which CO binding to its heme prosthetic group triggers a conformational change of CooA that allows the protein to bind its cognate target DNA sequence. By a powerful in vivo screening method following the simultaneous randomization of the codons for two C-helix residues, 113 and 116, near the distal heme pocket of CooA, we have isolated a series of novel CooA variants. In vivo, these show very high CO-independent activities (comparable with that of wild-type CooA in the presence of CO) and diminished CO-dependent activities. Sequence analysis showed that this group of variants commonly contains lysine at position 116 with a variety of residues at position 113. DNA-binding analysis of a representative purified variant, L116K CooA, revealed that this protein is competent to bind target DNA with K(d) values of 56 nm for Fe(III), 36 nm for Fe(II), and 121 nm for Fe(II)-CO CooA forms. Electron paramagnetic resonance and electronic absorption spectroscopies, combined with additional mutagenic studies, showed that L116K CooA has a new ligand replacing Pro(2) in both Fe(III) and Fe(II) states. The most plausible replacement ligand is the substituted lysine at position 116, so that the ligands of Fe(III) L116K CooA are Cys(75) and Lys(116) and those in the Fe(II) form are His(77) and Lys(116). A possible explanation for CO-independent activity in L116K CooA is that ligation of Lys(116) results in a repositioning of the C-helices at the CooA dimer interface. This result is consistent with that repositioning being an important aspect of the activation of wild-type CooA by CO.