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In this study, the first crystal structure of a novel crystal form of human insulin bound tometa-cresol in an acidic environment is reported. The combination of single-crystal and powder X-ray diffraction crystallography led to the detection of a previously unknown monoclinic phase (P21). The structure was identified from the powder patterns and was solved using single-crystal diffraction data at 2.2 Å resolution. The unit-cell parameters at pH 6.1 area= 47.66,b = 70.36,c = 84.75 Å, β = 105.21°. The structure consists of two insulin hexamers per asymmetric unit. The potential use of this insulin form in microcrystalline drugs is discussed.

This study focuses on the effects of the organic ligand 4-ethylresorcinol on the crystal symmetry and lattice dimensions of human insulin using powder X-ray crystallography., This study focuses on the effects of the organic ligand 4-ethylresorcinol on the crystal structure of human insulin using powder X-ray crystallography. For this purpose, systematic crystallization experiments have been conducted in the presence of the organic ligand and zinc ions within the pH range 4.50–8.20, while observing crystallization behaviour around the isoelectric point of insulin. High-throughput crystal screening was performed using a laboratory X-ray diffraction system. The most representative samples were selected for synchrotron X-ray diffraction measurements, which took place at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS). Four different crystalline polymorphs have been identified. Among these, two new phases with monoclinic symmetry have been found, which are targets for the future development of microcrystalline insulin drugs.

The primary focus of the present work is the study of the effects that two ligands and the crystallization pH have on the crystalline forms of human insulin. For this purpose, human insulin (HI) was co-crystallized with two distinct phenolic derivatives: the organic ligandsmeta-cresol (m-cresol) and 4-nitrophenol. The formation of polycrystalline precipitates was then followed by means of structural characterization of the individual specimens in terms of unit-cell symmetry and parameters. In both cases, two different polymorphs were identifiedviaX-ray powder diffraction measurements, the first of hexagonal symmetry (R3 space group) at higher pH values and the second of monoclinic symmetry (space groupP21) with unit-cell parametersa= 87.4282 (5),b = 70.5020 (3),c= 48.3180 (4) Å, β = 106.8958 (4)°, the latter of which to our knowledge has never been observed before.

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.

The effects of the ligands phenol and resorcinol on the crystallization of human insulin have been investigated as a function of pH. Powder diffraction data were used to characterize several distinct polymorphic forms. A previously unknown polymorph with monoclinic symmetry (P21) was identified for both types of ligand with similar characteristics [the unit-cell parameters for the insulin–resorcinol complex werea= 114.0228 (8),b= 335.43 (3),c= 49.211 (6) Å, β = 101.531 (8)°].

A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.

Comparisons of transcriptional and translational expression in normal and abnormal states are important to reach an understanding of pathogenesis and pathophysiology. Maintaining the biochemical, molecular, and structural sample integrity is essential for correct sample comparisons. We demonstrate that both proteins and neuropeptides, including their PTMs, are subjected to massive degradation in the brain already 1 min postmortem. Further, markers for determining the integrity and status of a biological sample were identified. The protein fragment stathmin 2-20 correlated well with the general level of postmortem degradation and may serve as a sample quality indicator for future work, both in animal and human postmortem brains. Finally, a novel method for preventing degradation of proteins and peptides in postmortem tissue is presented using rapid and uniform conductive heat transfer on tissue prior to the actual sample preparation procedures, which enables the relatively low-abundant neuropeptides to remain intact, minimizes degradation of proteins by proteolysis, and conserves the PTMs of the neuropeptides.

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 1.5A 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 2.2A 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.

A new database, SwePep, specifically designed for endogenous peptides, has been constructed to significantly speed up the identification process from complex tissue samples utilizing mass spectrometry. In the identification process the experimental peptide masses are compared with the peptide masses stored in the database both with and without possible post-translational modifications. This intermediate identification step is fast and singles out peptides that are potential endogenous peptides and can later be confirmed with tandem mass spectrometry data. Successful applications of this methodology are presented. The SwePep database is a relational database developed using MySql and Java. The database contains 4180 annotated endogenous peptides from different tissues originating from 394 different species as well as 50 novel peptides from brain tissue identified in our laboratory. Information about the peptides, including mass, isoelectric point, sequence, and precursor protein, is also stored in the database. This new approach holds great potential for removing the bottleneck that occurs during the identification process in the field of peptidomics. The SwePep database is available to the public.

Twelve different microcrystalline insulin formulations were investigated by X-ray powder diffraction and were shown to have very characteristic patterns. Three of the formulations crystallize in the same crystal system, but have structural differences in the N-terminal B-chain of the insulin molecule. This difference was efficiently detected in the powder patterns. The sensitivity of the method makes it a valuable tool for characterization of microcrystalline samples. By use of principal-component analysis, the twelve different formulations originating from six different crystal systems were classified into nine separate clusters. The powder patterns of each cluster can now be used as `fingerprints' for the different insulin polymorphs. The combination of X-ray powder diffraction and multivariate analysis, such as principal-component analysis, provides a rapid and effective tool for studying the influence of derivatives, additives, ions, pHetc., in the crystallization media.

Insulin degludec, an engineered acylated insulin, was recently reported to form a soluble depot after subcutaneous injection with a subsequent slow release of insulin and an ultralong glucose-lowering effect in excess of 40 h in humans. We describe the structure, ligand binding properties, and self-assemblies of insulin degludec using orthogonal structural methods. The protein fold adopted by insulin degludec is very similar to that of human insulin. Hexamers in the R(6) state similar to those of human insulin are observed for insulin degludec in the presence of zinc and resorcinol. However, under conditions comparable to the pharmaceutical formulation comprising zinc and phenol, insulin degludec forms finite dihexamers that are composed of hexamers in the T(3)R(3) state that interact to form an R(3)T(3)-T(3)R(3) structure. When the phenolic ligand is depleted and the solvent condition thereby mimics that of the injection site, the quaternary structure changes from dihexamers to a supramolecular structure composed of linear arrays of hundreds of hexamers in the T(6) state and an average molar mass, M(0), of 59.7 × 10(3) kg/mol. This novel concept of self-assemblies of insulin controlled by zinc and phenol provides the basis for the slow action profile of insulin degludec. To the best of our knowledge, this report for the first time describes a tight linkage between quaternary insulin structures of hexamers, dihexamers, and multihexamers and their allosteric state and its origin in the inherent propensity of the insulin hexamer for allosteric half-site reactivity.

A series of bovine insulin samples were obtained as 14 polycrystalline precipitates at room temperature in the pH range 5.0–7.6. High-resolution powder X-ray diffraction data were collected to reveal the T6 hexameric insulin form. Sample homogeneity and reproducibility were verified by additional synchrotron measurements using an area detector. Pawley analyses of the powder patterns displayed pH- and radiation-induced anisotropic lattice modifications. The pronounced anisotropic lattice variations observed for T6 insulin were exploited in a 14-data-set Rietveld refinement to obtain an average crystal structure over the pH range investigated. Only the protein atoms of the known structure with PDB code 2a3g were employed in our starting model. A novel approach for refining protein structures using powder diffraction data is presented. In this approach, each amino acid is represented by a flexible rigid body (FRB). The FRB model requires a significantly smaller number of refinable parameters and restraints than a fully free-atom refinement. A total of 1542 stereochemical restraints were imposed in order to refine the positions of 800 protein atoms, two Zn atoms and 44 water molecules in the asymmetric unit using experimental data in the resolution range 18.2–2.7 A for all profiles.

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.

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.

A novel set of protein descriptors has been developed to increase the understanding of protein behavior on chromatographic media. The protein descriptors are pH-dependent and based on electrostatic and hydrophobic properties of mainly the surface of the proteins as revealed by their three-dimensional structure. Interpretable and predictive quantitative structure property relationship (QSPR) models were then obtained for protein retention in ion exchange chromatography at different pH values. In most cases the calculated average surface potential could be directly related to retention times. Moreover, the high retention of human lactoferrin observed in cation exchange even at high pH values could be modeled by adding descriptors of the charge asymmetry.

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 C2221 and C2 respectively. The orthorhombic crystals were obtained at pH 6.5 and contained three insulin hexamers in R6 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 Å 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 C2221 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 Å in the C2221 crystals to 2.4 Å 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.