5 research outputs found
Dynamical origins of heat capacity changes in enzyme-catalysed reactions
Heat capacity changes affect the temperature dependence of enzyme catalysis, with implications for thermoadaptation, however their physical basis is unknown. Here the authors show that heat capacity changes are calculable by simulation, revealing distinct dynamical contributions from regions remote from the active site
Antimicrobial peptides within the Yellowtail Kingfish (Seriola lalandi)
A number of Seriola species are currently farmed or being investigated as future aquaculture species in countries around the world. However they face a number of issues and limitations which will need to be overcome to ensure future stability and growth, one of which are disease outbreaks. Despite this, very little has been done to understand the immune system of Seriola species and very few immune genes have been characterised. Antimicrobial peptides (AMP) are naturally occurring low molecular weight polypeptides that play a major role in an organism's immune system and act effectively as a first line of defence. This investigation isolates the full length cDNA sequences of two AMP's, piscidin and hepcidin from the yellowtail kingfish (Seriola lalandi). The full-length cDNA of the piscidin gene encodes a 65 amino acid prepropeptide, containing a 25-residue peptide, predicted to form an amphipathic helix-loop-helix structure. Phylogenetic analysis using fish piscidin sequences, showed that this AMP is only found in bony fish within the Acanthomorpha clade and that a possible three groups within the piscidin family exists, with S. lalandi belonging to a particular group. The full-length cDNA of the hepcidin gene encodes a 90 amino acid preprohepcidin, which contains a typical RX(R/K)R motif for cleavage of the mature peptide which comprises of eight conserved cysteine residues. Phylogenetic analysis of known vertebrate hepcidin antimicrobial peptide (HAMP) sequences, shows sequences from the Neoteleostei clade of bony fish form two very separate groups, HAMP1 and HAMP2, with the S. lalandi hepcidin gene grouped with the HAMP1 sequences. HAMP2 sequences are found to have multiple copies within fish and genome analysis showed very clearly that these two groups of genes are located on separate regions on the genome, with the multiple HAMP2 copies formed from tandem gene duplications. Lastly, using qPCR the expression of the S. lalandi piscidin gene within healthy fish was highest within, spleen and gills and lowest in liver, whereas hepcidin was highest in the liver with little or no expression in the spleen and gills.</p
Immunogenic fusion proteins induce neutralizing SARS-CoV-2 antibodies in the serum and milk of sheep
Antigen-specific polyclonal immunoglobulins derived from the serum, colostrum, or milk of immunized ruminant animals have potential as scalable therapeutics for the control of viral diseases including COVID-19. Here we show that the immunization of sheep with fusions of the SARS-CoV-2 receptor binding domain (RBD) to ovine IgG2a Fc domains promotes significantly higher levels of antigen-specific antibodies compared to native RBD or full-length spike antigens. This antibody population contained elevated levels of neutralizing antibodies that suppressed binding between the RBD and hACE2 receptors in vitro. A second immune-stimulating fusion candidate, Granulocyte-macrophage colony-stimulating factor (GM-CSF), induced high neutralizing responses in select animals but narrowly missed achieving significance. We further demonstrated that the antibodies induced by these fusion antigens were transferred into colostrum/milk and possessed cross-neutralizing activity against diverse SARS-CoV-2 variants. Our findings highlight a new pathway for recombinant antigen design in ruminant animals with applications in immune milk production and animal health
Cooperative Conformational Transitions Underpin the Activation Heat Capacity in the Temperature Dependence of Enzyme Catalysis
Many enzymes display non-Arrhenius behavior with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this behavior and recently the role of the activation heat capacity (ΔCP⧧) has been widely discussed. If enzyme-catalyzed reactions occur with appreciable negative values of ΔCP⧧ (arising from narrowing of the conformational space along the reaction coordinate), then curved Arrhenius plots are a consequence. To investigate these phenomena in detail, we have collected high precision temperature-rate data over a wide temperature interval for a model glycosidase enzyme MalL, and a series of mutants that change the temperature-dependence of the enzyme-catalyzed rate. We use these data to test a range of models including macromolecular rate theory (MMRT) and an equilibrium model. In addition, we have performed extensive molecular dynamics (MD) simulations to characterize the conformational landscape traversed by MalL in the enzyme-substrate complex and an enzyme-transition state complex. We have crystallized the enzyme in a transition state-like conformation in the absence of a ligand and determined an X-ray crystal structure at very high resolution (1.10 Å). We show (using simulation) that this enzyme-transition state conformation has a more restricted conformational landscape than the wildtype enzyme. We coin the term "transition state-like conformation (TLC)" to apply to this state of the enzyme. Together, these results imply a cooperative conformational transition between an enzyme-substrate conformation (ES) and a transition-state-like conformation (TLC) that precedes the chemical step. We present a two-state model as an extension of MMRT (MMRT-2S) that describes the data along with a convenient approximation with linear temperature dependence of the activation heat capacity (MMRT-1L) that can be used where fewer data points are available. Our model rationalizes disparate behavior seen for MalL and previous results for a thermophilic alcohol dehydrogenase and is consistent with a raft of data for other enzymes. Our model can be used to characterize the conformational changes required for enzyme catalysis and provides insights into the role of cooperative conformational changes in transition state stabilization that are accompanied by changes in heat capacity for the system along the reaction coordinate. TLCs are likely to be of wide importance in understanding the temperature dependence of enzyme activity and other aspects of enzyme catalysis.</p