Advancing predictions of protein stability in the solid state.

The β-relaxation associated with the sub-glass transition temperature (Tg,β) is attributed to fast, localised molecular motions which can occur below the primary glass transition temperature (Tg,α). Consistent with Tg,β being observed well-below storage temperatures, the β-relaxation associated motions have been hypothesised to influence protein stability in the solid state and could thus impact the quality of e.g. protein powders for inhalation or reconstitution and injection. Why then do distinct solid state protein formulations with similar aggregation profiles after drying and immediate reconstitution, display different profiles when reconstituted following prolonged storage? Is the value of Tg,β, associated with the β-relaxation process of the system, a reliable parameter for characterising the behaviour of proteins in the solid state? Bearing this in mind, in this work we further explore the different relaxation dynamics of glassy solid state monoclonal antibody formulations using terahertz time-domain spectroscopy and dynamical mechanical analysis. By conducting a 52 week stability study on a series of multi-component spray-dried formulations, an approach for characterising and analysing the solid state dynamics and how these relate to protein stability is outlined.EPSR

Tracking Solid State Dynamics in Spray-Dried Protein Powders at Infrared and Terahertz Frequencies

Therapeutic protein powders can be prepared by spray-drying. This process is known to result in solid particles of relatively narrow size distribution and high yield and purity [1], [2]. Additionally, the spray-drying process is rapid, semi-continuous, cost-effective, reproducible and scalable. The process transforms a liquid into dry particles by atomising the liquid feed in a hot drying gas stream [3]. One of the main advantages of spray-drying is that a wide range of formulations, including heat-sensitive materials, can be dried using this technique since the droplet surface will retain the wet-bulb temperature rather than the temperature of the hot drying gas, provided evaporation is taking place at the droplet surface. By the time the evaporation at the droplet/particle surface stops, the drying gas will already have cooled down, thus limiting the heat exposure of the formulation components to the relatively high inlet gas temperatures, and, in combination with the short process duration, making spray-drying a feasible process for heat-sensitive materials, including proteins [1], [2], [3]. While spray-drying is a well established process for small molecules, the additional challenge of ensuring protein stability of the dried product during storage currently limits its use for biopharmaceutical products [2], [4]. A major concern during the spray-drying process is the entire or partial unfolding of proteins due to their high susceptibility to migrate to the air-liquid interfaces where the surface energies can cause the protein to expose hydrophobic regions, resulting in facilitated protein-protein interactions and ultimately aggregation [5]. In order to prevent such undesired aggregation non-ionic surfactants, for example polysorbate, are often used to prevent accumulation of protein at the air-liquid interface, as these small and more mobile surfactants will preferentially position themselves at the interfaces [6]. To put more generally, the excipients of a formulation are vital in providing stability to the protein by maintaining its native conformation during the spray-drying process.T.A.S. and J.A.Z. acknowledge funding from AstraZeneca UK Limited/MedImmune Limited and the UK Engineering and Physical Sciences Research Council (EP/N022769/1). T.A.S. would like to thank the AJA-Karten Trust and the AIA-Kenneth Lindsay Trust for their financial support

Insights into the architecture and stoichiometry of Escherichia coli PepA•DNA complexes involved in transcriptional control and site-specific DNA recombination by atomic force microscopy

Multifunctional Aminopeptidase A (PepA) from Escherichia coli is involved in the control of two distinct DNA transaction processes: transcriptional repression of the carAB operon, encoding carbamoyl phosphate synthase and site-specific resolution of ColE1-type plasmid multimers. Both processes require communication at a distance along a DNA molecule and PepA is the major structural component of the nucleoprotein complexes that underlie this communication. Atomic Force Microscopy was used to analyze the architecture of PepA·carAB and PepA·cer site complexes. Contour length measurements, bending angle analyses and volume determinations demonstrate that the carP1 operator is foreshortened by ∼235 bp through wrapping around one PepA hexamer. The highly deformed part of the operator extends from slightly upstream of the –35 hexamer of the carP1 promoter to just downstream of the IHF-binding site, and comprises the binding sites for the PurR and RutR transcriptional regulators. This extreme remodeling of the carP1 control region provides a straightforward explanation for the strict requirement of PepA in the establishment of pyrimidine and purine-specific repression of carAB transcription. We further provide a direct physical proof that PepA is able to synapse two cer sites in direct repeat in a large interwrapped nucleoprotein complex, likely comprising two PepA hexamers

New Insight into the Transcarbamylase Family: The Structure of Putrescine Transcarbamylase, a Key Catalyst for Fermentative Utilization of Agmatine

Transcarbamylases reversibly transfer a carbamyl group from carbamylphosphate (CP) to an amine. Although aspartate transcarbamylase and ornithine transcarbamylase (OTC) are well characterized, little was known about putrescine transcarbamylase (PTC), the enzyme that generates CP for ATP production in the fermentative catabolism of agmatine. We demonstrate that PTC (from Enterococcus faecalis), in addition to using putrescine, can utilize L-ornithine as a poor substrate. Crystal structures at 2.5 Å and 2.0 Å resolutions of PTC bound to its respective bisubstrate analog inhibitors for putrescine and ornithine use, N-(phosphonoacetyl)-putrescine and δ-N-(phosphonoacetyl)-L-ornithine, shed light on PTC preference for putrescine. Except for a highly prominent C-terminal helix that projects away and embraces an adjacent subunit, PTC closely resembles OTCs, suggesting recent divergence of the two enzymes. Since differences between the respective 230 and SMG loops of PTC and OTC appeared to account for the differential preference of these enzymes for putrescine and ornithine, we engineered the 230-loop of PTC to make it to resemble the SMG loop of OTCs, increasing the activity with ornithine and greatly decreasing the activity with putrescine. We also examined the role of the C-terminal helix that appears a constant and exclusive PTC trait. The enzyme lacking this helix remained active but the PTC trimer stability appeared decreased, since some of the enzyme eluted as monomers from a gel filtration column. In addition, truncated PTC tended to aggregate to hexamers, as shown both chromatographically and by X-ray crystallography. Therefore, the extra C-terminal helix plays a dual role: it stabilizes the PTC trimer and, by shielding helix 1 of an adjacent subunit, it prevents the supratrimeric oligomerizations of obscure significance observed with some OTCs. Guided by the structural data we identify signature traits that permit easy and unambiguous annotation of PTC sequences

Nurses' perceptions of aids and obstacles to the provision of optimal end of life care in ICU

Contains fulltext : 172380.pdf (publisher's version ) (Open Access

Metabolic channelling of carbamoyl phosphate in the hyperthermophilic archaeon Pyrococcus furiosus: dynamic enzyme-enzyme interactions involved in the formation of the channelling complex

Protection of thermolabile metabolites and coenzymes is a somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles. Detailed information about the mechanisms used by thermophiles to protect these thermolabile metabolites and coenzymes is still scarce. A case in point is CP (carbamoyl phosphate), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate. Recently we obtained the first evidence for a physical interaction between two hyperthermophilic enzymes for which kinetic evidence had suggested that these enzymes channel a highly thermolabile and potentially toxic intermediate. By physically interacting with each other, CKase (carbamate kinase) and OTCase (ornithine carbarnoyltransferase) prevent thermodenaturation of CP in the aqueous cytoplasmic environment. The CP channelling complex involving CKase and OTCase or ATCase (aspartate carbarnoyltransferase), identified in hyperthermophilic archaea, provides a good model system to investigate the mechanism of metabolic channelling and the molecular basis of pfotein-protein interactions in the physiology of extreme thermophiles

Tracking solid state dynamics in spray-dried protein powders at infrared and terahertz frequencies

status: publishe

Mechanism of thermal decomposition of carbamoyl phosphate and its stabilization by aspartate and ornithine transcarbamoylases

Carbamoyl phosphate (CP) has a half-life for thermal decomposition of <2 s at 100 °C, yet this critical metabolic intermediate is found even in organisms that grow at 95–100 °C. We show here that the binding of CP to the enzymes aspartate and ornithine transcarbamoylase reduces the rate of thermal decomposition of CP by a factor of >5,000. Both of these transcarbamoylases use an ordered-binding mechanism in which CP binds first, allowing the formation of an enzyme·CP complex. To understand how the enzyme·CP complex is able to stabilize CP we investigated the mechanism of the thermal decomposition of CP in aqueous solution in the absence and presence of enzyme. By quantum mechanics/molecular mechanics calculations we show that the critical step in the thermal decomposition of CP in aqueous solution, in the absence of enzyme, involves the breaking of the CO bond facilitated by intramolecular proton transfer from the amine to the phosphate. Furthermore, we demonstrate that the binding of CP to the active sites of these enzymes significantly inhibits this process by restricting the accessible conformations of the bound ligand to those disfavoring the reactive geometry. These results not only provide insight into the reaction pathways for the thermal decomposition of free CP in an aqueous solution but also show why these reaction pathways are not accessible when the metabolite is bound to the active sites of these transcarbamoylases