Virus-like particles (VLPs) have been extensively explored as nanoparticle vehicles for many applications in biotechnology (e.g., vaccines, drug delivery, imaging agents, biocatalysts). However, amino acid sequence plasticity relative to subunit expression and nanoparticle assembly has not been explored. Whereas the hepatitis B core protein (HBc) VLP appears to be the most promising model for fundamental and applied studies; particle instability, antigen fusion limitations, and intrinsic immunogenicity have limited its development. Here, we apply Escherichia coli-based cell-free protein synthesis (CFPS) to rapidly produce and screen HBc protein variants that still self-assemble into VLPs. To improve nanoparticle stability, artificial covalent disulfide bridges were introduced throughout the VLP. Negative charges on the HBc VLP surface were then reduced to improve surface conjugation. However, removal of surface negative charges caused low subunit solubility and poor VLP assembly. Solubility and assembly as well as surface conjugation were greatly improved by transplanting a rare spike region onto the common shell structure. The newly stabilized and extensively modified HBc VLP had almost no immunogenicity in mice, demonstrating great promise for medical applications. This study introduces a general paradigm for functional improvement of complex protein assemblies such as VLPs. This is the first study, to our knowledge, to systematically explore the sequence plasticity of viral capsids as an approach to defining structure function relationships for viral capsid proteins. Our observations on the unexpected importance of the HBc spike tip charged state may also suggest new mechanistic routes toward viral therapeutics that block capsid assembly.virus-like particle | engineered nanoparticles | disulfide stabilization | hepatitis core protein | cell-free protein synthesis V irus-like particles (VLPs) are probably the most precisely defined and, therefore, potentially the most useful complex nanometer-scale scaffolds (1). VLPs mimic the capsid structure of real viruses, but lack infectious genetic material. Selected VLPs derived from pathogens have already provided major advances in the development of vaccines that have known and relatively homogeneous structures as well as enhanced immunogenicity (2). Such nanoparticles provide comparable cellular uptake and intracellular trafficking compared with natural viruses (3), and also have repetitive surfaces for the high-density display of vaccine antigens (4). In addition, VLPs offer favorable trafficking from the injection site to lymph nodes (5). Since the first reported use of a hepatitis B core protein (HBc) VLP as an antigen carrier in 1987 (6), at least 110 VLP vaccine candidates have been constructed by using capsid proteins from 35 different viral families (7).Among different types of VLPs, the HBc VLP is the most flexible and promising model for fundamental and applied immunological studies (8). One advantage of the HBc VLP platform is that the capsids can be produ...

1 The eects of adenosine and adenine on the gating of native sheep cardiac ryanodine receptor (RyR) channels were investigated. By examining the mechanisms underlying channel activation and by using comparative molecular ®eld analysis (CoMFA) we have investigated the structural features of adenine-based ligands involved in channel activation. 2 In the presence of 10 mM cytosolic Ca 2+ , adenosine and adenine both activate the channel but only to a level approximately 10 and 20% respectively of that of ATP indicating that both are partial agonists of low ecacy. 3 Adenosine was able to antagonize the ATP-induced increase in open probability (Po) as expected for a partial agonist of low ecacy at the ATP sites on the cardiac RyR. 4 GTP (100 mM ± 10 mM) had no eect on channel gating indicating that the adenine ring structure is important for agonist activity at the ATP-sites on RyR. 5 CoMFA revealed an extremely strong correlation between the structural features of the ®ve ATP analogues and the ability to increase (Po). Our model indicates that the high ecacy of ATP results primarily from the large electrostatic ®eld established by the ionized phosphate groups. Reducing the number of phosphate groups lowers the strength of this ®eld, leading to ligands with lower ecacy. In addition, steric interactions between the a-phosphate and ribose moieties and the RyR are correlated with low Po.

Bacterial flagellin has been explored as a potential vaccine adjuvant for enhancing immune responses. In this article, we describe Escherichia coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce soluble phase 1 flagellin (FliC) protein from Salmonella typhimurium. The yield was about 300 µg/mL and the product had much higher affinity for the TLR5 receptor (EC50 = 2.4 ± 1.4 pM) than previously reported. The flagellin coding sequence was first optimized for cell-free expression. We then found that the D0 domain at the C-terminus of flagellin was susceptible to proteolytic degradation in the CFPS system. Proteolysis was reduced by protease inhibitors, the use of protease-deficient cell extracts or deletion of the flagellin D0 domain. A human Toll-Like Receptor 5 (hTLR5)-specific bioactivity analysis of purified flagellin demonstrated that, although the D0 domain is far from the TLR5 recognition region, it is important for flagellin bioactivity. We next incorporated a non-natural amino acid displaying an alkyne moiety into flagellin using the CFPS system and attached flagellin to hepatitis B core virus-like particles (VLPs) using bioorthogonal azide-alkyne cycloaddition reactions. The ordered and oriented VLP display of flagellin increased its specific TLR5 stimulation activity by approximately 10-fold.

Malaria, one of the most common vector borne human diseases, is a major world health issue. In 2015 alone, more than 200 million people were infected with malaria, out of which, 429 000 died. Even though artemisinin-based combination therapies (ACT) are highly effective at treating malaria infections, novel efforts toward development of vaccines to prevent transmission are still needed. Pfs25, a postfertilization stage parasite surface antigen, is a leading transmission-blocking vaccine (TBV) candidate. It is postulated that Pfs25 anchors to the cell membrane using a glycosylphosphatidylinositol (GPI) linker, which itself possesses pro-inflammatory properties. In this study, Escherichia coli derived extract (XtractCF+TM) was used in cell free protein synthesis [CFPS] to successfully express >200 mg/L of recombinant Pfs25 with a C-terminal non-natural amino acid (nnAA), namely, p-azidomethyl phenylalanine (pAMF), which possesses a reactive azide group. Thereafter, a unique conjugate vaccine (CV), namely, Pfs25-GPI was generated with dibenzocyclooctyne (DBCO) derivatized glycan core of malaria GPI using a simple but highly efficient copper free click chemistry reaction. In mice immunized with Pfs25 or Pfs25-GPI, the Pfs25-GPI group showed significantly higher titers compared to the Pfs25 group. Moreover, only purified IgGs from Pfs25-GPI group were able to significantly block transmission of parasites to mosquitoes, as judged by a standard membrane feeding assay [SMFA]. To our knowledge, this is the first report of the generation of a CV using Pfs25 and malaria specific GPI where the GPI is shown to enhance the ability of Pfs25 to elicit transmission blocking antibodies.

Q H]ryanodine binding and increasing the Po of the channels was FDP s glucose-1-phosphate (G-1-P) s fructose-6-phosphate (F-6-P) s glucose-6-phosphate (G-6-P). These novel properties of the sugar phosphate compounds indicate that changes in glycolytic flux may influence the release of SR Ca P+ by modulating RyR channel gating. z 1998 Federation of European Biochemical Societies.

Comparative molecular field analysis (CoMFA) predicts that the large electrostatic field around the phosphate groups of ATP plays a crucial role in stabilizing the open state of the cardiac ryanodine receptor (RyR) channel. We therefore investigated the effects of adenosine-5Ј-(␤,␥-methylenetriphosphate) (AMP-PCP), an ATP analog with lower negative charge in this region, on the gating of the cardiac RyR channel. In the presence of 10 M cytosolic Ca 2ϩ , AMP-PCP exhibited approximately 50% of the efficacy of ATP and optimal doses increased open probability (P o ) to only 0.441 Ϯ 0.156 (n ϭ 4), thus confirming the predictive ability of our preliminary CoMFA model. We also reveal that AMP-PCP has a higher affinity than ATP for the cardiac RyR, demonstrating that the structural properties required for tight binding to RyR differ from those necessary for recruiting long open states and high P o values. CoMFA identified very strong correlations between the structures of adeninebased ligands and their affinity for RyR and different (but also highly significant) correlations between structure and the ability to activate the channel. Analysis indicates that ATP may be more effective than other adenine nucleotides because it can convert the greatest amount of binding energy into conformational changes that stabilize the open channel state. (Kermode et al., 1998). High levels of adenine nucleotides and Ca 2ϩ tend to inactivate the channel (Kermode et al., 1998;Ching et al., 1999); therefore, the effects of this group of ligands are complex and are likely to shape both the activation and inactivation processes of intracellular Ca 2ϩ release in cardiac cells. Understanding the underlying molecular nature of the binding of adenine nucleotides to RyR channels and the structural features of ATP that produce open RyR channels is therefore important for a greater understanding of how RyR channels are regulated during the process of excitation-contraction coupling. In a preliminary CoMFA study, we previously correlated the structure of ATP and other adenine-based ligands with the ability to modulate the gating of native sheep cardiac RyR incorporated into planar phospholipid bilayers (Chan et al., 2000). With millimolar luminal [Ca 2ϩ ] and a maintained cytosolic [Ca 2ϩ ] of 10 M, ATP induced P o levels of approximately 0.9. The maximum P o levels produced by the other adenine-based ligands investigated (ADP, AMP, adenosine, adenine) were much lower. CoMFA demonstrated a high correlation between ligand structure and maximum P o induced. Although it is now well established that the purine ring is important for agonist activity (Morii and Tonomura, 1983;Meissner, 1984;Chan et al., 2000), we demonstrated that the phosphate groups are essential to the high efficacy of ATP. Our model predicts that the charge produced by the phosphate groups is the single most important factor that enables bound ATP molecules to activate the cardiac RyR channel to a greater extent than ligands with fewer phosphate groups (for example, ADP a...

ATP is an essential constitutive regulator of cardiac ryanodine receptors (RyR2), enabling small changes in cytosolic Ca2+ to trigger large changes in channel activity. With recent landmark determinations of the full structures of RyR1 (skeletal isoform) and RyR2 using cryo-EM, and identification of the RyR1 ATP binding site, we have taken the opportunity to model the binding of fragments of ATP into RyR2 in order to investigate how the structure of the ATP site dictates the functional responses of ligands attracted there. RyR2 channel gating was assessed under voltage-clamp conditions and by [3H]ryanodine binding studies. We show that even the triphosphate (PPPi) moiety alone was capable of activating RyR2 but produced two distinct effects (activation or irreversible inactivation) that we suggest correspond to two preferred binding locations within the ATP site. Combinations of complementary fragments of ATP (Pi + ADP or PPi + AMP) could not reproduce the effects of ATP, however, the presence of adenosine prevented the inactivating PPPi effects, allowing activation similar to that of ATP. RyR2 appears to accommodate diverse types of molecules, including PPPi, deep within the ATP binding site. The most effective ligands, however, have at least three phosphate groups that are guided into place by a nucleoside.

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