Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH

Background: Insulin is a therapeutic protein that is widely used for the treatment of diabetes. Its biological function was discovered more than 80 years ago and it has since then been characterized extensively. Crystallization of the insulin molecule has always been a key activity since the protein is often administered by subcutaneous injections of crystalline insulin formulations. Over the years, insulin has been crystallized and characterized in a number of crystal systems. Results: Interestingly, we have now discovered two new crystal forms of human insulin. The crystals were obtained when the two chaotropic agents, urea and thiocyanate were present in the crystallization experiments, and their structures were determined by X-ray crystallography. The crystals belong to the orthorhombic and monoclinic crystal systems, with space groups C222(I) and C2 respectively. The orthorhombic crystals were obtained at pH 6.5 and contained three insulin hexamers in R-6 conformation in the asymmetric unit whilst the monoclinic C2 crystals were obtained at pH 7.0 and contained one R6 hexamer in the asymmetric unit. Common for the two new crystals is a hexamer-hexamer interaction that has not been found in any of the previous crystal forms of insulin. The contacts involve a tight glutamate-glutamate interaction with a distance of 2.3 angstrom between groups. The short distance suggests a low barrier hydrogen bond. In addition, two tyrosine-tyrosine interactions occupying a known phenol binding pocket contribute to the stabilization of the contacts. Within the crystals, distinct binding sites for urea were found, adding further to the discussion on the role of urea in protein denaturation. Conclusion: The change in space group from C222(I) to C2 was primarily caused by an increase in pH. The fewer number of hexamer-hexamer interactions comprising the short hydrogen bond in the C2 space group suggest that pH is the driving force. In addition, the distance between the two glutamates increases from 2.32 angstrom in the C222(I) crystals to 2.4 angstrom in the C2 crystals. However, in both cases the low barrier hydrogen bond and the tyrosine-tyrosine interaction should contribute to the stability of the crystals which is crucial when used in pharmaceutical formulations

Developing adaptive capacity in times of climate change in central rural Vietnam: exploring smallholders’ learning and governance

Climate change already affects Vietnam in virtually all sectors. Agriculture in small communities is particularly vulnerable to current and projected climate change impacts. Many of the smallholder farmers in Vietnam have limited adaptive capacity to deal with these impacts. Increasingly social learning is proposed as an important mechanism to build the adaptive capacity of local farming communities. However, little is known about the interplay between social learning and adaptive capacity and how adaptive capacity could be increased in a complex hierarchical governance setting that is typical in a country like Vietnam. The dissertation therefore aims to elicit and explore the ways through which social learning can increase the adaptive capacity of smallholder farmers in central Vietnam to respond to climate change impacts. Four research questions are addressed: (i) what insights does the existing body of climate change adaptation literature provide into the interplay between social learning and adaptive capacity?; (ii) what do smallholder farmers in Vietnam perceive as their current adaptive capacity and what enables or constrains them in increasing it?; (iii) how can social learning configurations strengthen the adaptive capacity of farming communities?; and (iv) how do different levels of government enable and constrain the process of building adaptive capacity and social learning of smallholder farmers to respond to impacts of climate change in Vietnam? Overall, the dissertation shows that social learning offers many possibilities to help farmers adapt to climate change, but that climate change adaptation in developing countries creates specific contextual conditions that require an adaptive capacity-focused perspective. An adequate learning configuration that can successfully help farmers build their adaptive capacity, considers responsive design, facilitation, monitoring, and evaluation steps. Furthermore, efforts of increasing adaptive capacity should not only focus on technical, social and human dimensions, but also on market conditions. The critical importance in creating an environment that enables social learning is the role of government across different levels. In order for the Vietnamese government to be more actively involved in building adaptive capacity through social learning, investments in transparent legal institutions, efficient use of limited available resources, and enhancing capacity of local policy actors will be critical in helping smallholder farmers learn how to adapt to climate change impacts.</p

A bispecific antibody approach for the potential prophylactic treatment of inherited bleeding disorders

Inherited bleeding disorders such as Glanzmann thrombasthenia (GT) lack prophylactic treatment options. As a result, serious bleeding episodes are treated acutely with blood product transfusions or frequent, repeated intravenous administration of recombinant activated coagulation factor VII (rFVIIa). Here we describe HMB-001, a bispecific antibody designed to bind and accumulate endogenous FVIIa and deliver it to sites of vascular injury by targeting it to the TREM (triggering receptor expressed on myeloid cells)-like transcript-1 (TLT-1) receptor that is selectively expressed on activated platelets. In healthy nonhuman primates, HMB-001 prolonged the half-life of endogenous FVIIa, resulting in its accumulation. Mouse bleeding studies confirmed antibody-mediated potentiation of FVIIa hemostatic activity by TLT-1 targeting. In ex vivo models of GT, HMB-001 localized FVIIa on activated platelets and potentiated fibrin-dependent platelet aggregation. Taken together, these results indicate that HMB-001 has the potential to offer subcutaneous prophylactic treatment to prevent bleeds in people with GT and other inherited bleeding disorders, with a low-frequency dosing regimen

Structural characterization of insulin NPH formulations

Insulin NPH (neutral protamine hagedorn) has for long been one of the most important therapeutic formulations for the treatment of diabetes. The protracted action profile of NPH formulations is gained from crystallizing insulin with zinc in the presence of the basic poly-arginine peptide protamine. In spite of its long history and successful use, the binding mode of the insulin-protamine complex is not known. in this study, three different systems were used to study protamine binding to insulin. In the first system, crystals of an insulin-protamine complex grown in the presence of urea and diffracting to 1.5 angstrom resolution were analyzed. In the second system, a shorter peptide consisting of 12 arginine residues was co-crystallized with insulin in order to reduce the flexibility and thereby improve the electron density of the peptide. Both systems yielded data to a significantly higher resolution than obtained previously. In addition, a third system was analyzed where crystals of insulin and protamine were grown in the absence of urea, with conditions closely resembling the pharmaceutical formulation. Data from these NPH microcrystals could for the first time be collected to 2.2 angstrom resolution at a micro focused X-ray beamline. Analysis of all three crystal forms reveal potential protamine density located close to the solvent channel leading to the centrally located zinc atoms in the insulin hexamer and support that protamine binds to insulin in a not well defined conformation. (c) 2007 Elsevier B.V. All rights reserved

Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH-4

<p><b>Copyright information:</b></p><p>Taken from "Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH"</p><p>http://www.biomedcentral.com/1472-6807/7/83</p><p>BMC Structural Biology 2007;7():83-83.</p><p>Published online 19 Dec 2007</p><p>PMCID:PMC2241603.</p><p></p>shown to bind in the pocket created by the two flanking tyrosine residues. The side chain of the tyrosine to the right in is missing in the pdb file. In (b) the same structures are superposed with the C222urea structure (orange). The side chain of the left tyrosine is flipped to accommodate the hexamer-hexamer interaction shown in (c), where a neighboring hexamer from the asymmetric unit is included (grey). The tyrosine side chain of the second hexamer occupies the same position as the phenolic compounds

Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH-0

<p><b>Copyright information:</b></p><p>Taken from "Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH"</p><p>http://www.biomedcentral.com/1472-6807/7/83</p><p>BMC Structural Biology 2007;7():83-83.</p><p>Published online 19 Dec 2007</p><p>PMCID:PMC2241603.</p><p></p>The flanking hexamers are located around the central hexamer at an angle of ~110°. The local non-crystallographic three-fold axis of the two outer hexamers is almost orthogonal to the central non-crystallographic three-fold axis. The zinc atoms are illustrated as large spheres to mark the position of the three-fold axes. The hexamers are numbered from I to III. (b) The crystal packing in the C222space group drawn with main chain trace with the asymmetric unit in magenta. (c) The crystal packing of the human insulin in space group C2. The asymmetric unit molecule is colored magenta. The inserts in (b) and (c) show crystals of the C222and C2 forms, respectively

Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH-5

<p><b>Copyright information:</b></p><p>Taken from "Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH"</p><p>http://www.biomedcentral.com/1472-6807/7/83</p><p>BMC Structural Biology 2007;7():83-83.</p><p>Published online 19 Dec 2007</p><p>PMCID:PMC2241603.</p><p></p>rily directed towards the carbonyl oxygen GlnA5 but surrounding carbonyl oxygens from SerA9 and IleA10 are within reasonable distances. Marked distances are given in Ångström (Å)

Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH-1

<p><b>Copyright information:</b></p><p>Taken from "Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH"</p><p>http://www.biomedcentral.com/1472-6807/7/83</p><p>BMC Structural Biology 2007;7():83-83.</p><p>Published online 19 Dec 2007</p><p>PMCID:PMC2241603.</p><p></p>B chains in the C222structures have the PheB1 residue in an extended conformation (the top most population). Labels indicate chain names used in the final PDB files. For illustrative purpose, the side chain of the C-terminal LysB29 is included in the figures to illustrate the flexibility. This side-chain was subsequently omitted from several chains in the final PDB files due to disordered electron density