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

Engineering of Insulin Receptor Isoform-Selective Insulin Analogues

BACKGROUND: The insulin receptor (IR) exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues. METHODOLOGY/PRINCIPAL FINDINGS: Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference) even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity. CONCLUSIONS/SIGNIFICANCE: We have discovered a new class of IR isoform-selective insulin analogues with 2-4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits

Insulin Polymorphism Crystallographic Characterization of Insulin Microcrystals

Insulin is a protein needed for the uptake of glucose from the circulating blood. In absence of sufficiently high insulin levels in the body or when the cells have a reduced sensitivity for insulin a disease state referred to as diabetes occurs. Treatment of diabetes generally requires daily injections of exogenous insulin. Many of the pharmaceutical formulations consist of insulin in a crystalline state. After injection, the crystals start to dissolve and the insulin molecules diffuse into the bloodstream. Crystal size, morphology and crystal packing affect the action profile of the formulation. Other factors with impact on the duration include additives and ligands such as zinc and phenolic molecules. A careful characterization of the microcrystals is important both from a research perspective but also for regulatory reasons. Within this project, X-ray crystallography was used to characterize microcrystals of insulin. The small size of the crystals makes visual interpretation difficult, and determination of crystal form based on crystal morphology is sometimes not possible. General single crystal X-ray analysis is of limited use for characterization of the microcrystals since the crystals are too small. X-ray powder diffraction was therefore utilized. Several suspensions of insulin microcrystals were characterized by this method. Insulin is a polymorphic protein that can be crystallized in a number of different crystal forms and space groups. It was shown that the different crystal forms had specific X-ray powder patterns. The patterns could thus serve as fingerprints for certain crystal forms. Both pharmaceutical formulations and crystalline suspension from research activities could be characterized. Polymorphism within samples could be detected and even two new crystal forms of insulin were identified. For efficient analysis of the powder patterns a multivariate analysis method was used, principal component analysis (PCA). This facilitated analysis and visualization of the data considerably. One of the new crystal forms was subsequently structurally determined by single crystal analysis after modification of the crystallization conditions to promote growth of larger crystals. The space group was orthorhombic C2221. The asymmetric unit contained three hexamers with a novel crystal packing between hexamers. By increasing the pH by ~0.5 pH units to 7.0 a second new crystal form was found (monoclinic C2). The major difference was a fewer number of the novel crystal packing interactions between the hexamers in this crystal. The packing contact involves two tyrosine-tyrosine interactions and a tight glutamate-glutamate interaction of 2.4 Å. When pH was increased further (above 7.0) the dominant crystal form was the previously well characterized monoclinic P21, with no crystal packing interactions of this kind. The powder diffraction methods utilized in this project was useful for determination of crystal form of the microcrystals. As a complement, it was shown that the microcrystals could be used to solve the structure by using a microfocused X-ray beam at a designated beamline. The structure of human insulin was solved at a resolution of 2.2 Å, from orthorhombic crystals in space group P212121. The human insulin was co-crystallized with the peptide protamine consisting mainly of arginine residues. Such crystals have long been used for the treatment of diabetes and are referred to as NPH crystals (neutral protamine Hagedorn). Due to crystallographic disorder of the peptide, the protamine could however not be identified in the electron density map. The disorder suggests that the insulin-protamine interaction is unspecific and that the primary function of the protamine is to balance the overall charge of insulin

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-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

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-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-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