Phenol stabilizes more helix in a new symmetrical zinc insulin hexamer

Derewenda, Urszula; Derewenda, Zygmunt S.; Dodson, E.J.; Dodson, Guy; Reynolds, C. D.; Smith, G. D.; Sparks, C.A.; Swenson, Dale C.

doi:10.1038/338594a0

Cited by 338 publications

(444 citation statements)

References 12 publications

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“…Strikingly similar fragments have been located in crystal interfaces of insulin in drug formulations containing the protamine clupeine (Balschmidt et al, 1991). Interestingly, in this insulin structure, a phenol derivative (m-cresol) has also been found to mediate crystal contacts, and phenol has been used to stabilize other insulins (Derewenda et al, 1989). In the absence of a clear assignment, we have modeled our density as a three-and a 16-residue polyglycine backbone (possibly led by an N-terminal lysine residue) as a placeholder for what is probably a mixture of intercalated peptides and other SPE components.…”

Section: Packing Analysis Of Trypsinmentioning

confidence: 89%

Operator-assisted harvesting of protein crystals using a universal micromanipulation robot

et al. 2007

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High-throughput crystallography has reached a level of automation where complete computer-assisted robotic crystallization pipelines are capable of cocktail preparation, crystallization plate setup, and inspection and interpretation of results. While mounting of crystal pins, data collection and structure solution are highly automated, crystal harvesting and cryocooling remain formidable challenges towards full automation. To address the final frontier in achieving fully automated high-throughput crystallography, the prototype of an anthropomorphic six-axis universal micromanipulation robot (UMR) has been designed and tested; this UMR is capable of operator-assisted harvesting and cryoquenching of protein crystals as small as 10 mm from a variety of 96-well plates. The UMR is equipped with a versatile tool exchanger providing full operational flexibility. Trypsin crystals harvested and cryoquenched using the UMR have yielded a 1.5 Å structure demonstrating the feasibility of robotic protein crystal harvesting.

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Section: Packing Analysis Of Trypsinmentioning

confidence: 89%

Operator-assisted harvesting of protein crystals using a universal micromanipulation robot

et al. 2007

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“…In the hexameric form the HI molecule exists in two distinct conformations called the T-state and the R-state deviating in the length of the central a-helix in the B-chain. 2,6,7 In the R-state there is a continuous a-helix from residue B1 to B19, whilst in the T-state the first eight residues are found in an extended conformation.…”

Section: Introductionmentioning

confidence: 99%

“…The hexameric form can be further stabilized by addition of small aromatic alcohols (phenol, m-cresol) and salt which further stabilizes the R-state. 7 Despite the stabilisation through formulation there is still a desire to further increase insulin's stability. Such insulin analogs would likely have longer in use time, not require refrigeration and could be used in continuous insulin pumps.…”

Section: Introductionmentioning

confidence: 99%

Insulin analog with additional disulfide bond has increased stability and preserved activity

et al. 2013

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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 Ca-Ca 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 Bchain 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.

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“…phenol to the crystallization medium also results in the formation of an cc-helix in the B1 -B8 region [8].…”

Section: -___mentioning

confidence: 99%

The solution structure of a monomeric insulin

Knegtel

Boelens

Ganadu

et al. 1991

European Journal of Biochemistry

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The solution conformation of des-(B26-B30)-insulin (DPI) has been investigated by 'H-NMR spectroscopy. A set of 250 approximate interproton distance restraints, derived from two-dimensional nuclear Overhauser enhancement spectra, were used as the basis of a structure determination using distance geometry (DG) and distance-bound driven dynamics (DDD). Sixteen DG structures were optimized using energy minimization (EM) and submitted to short 5-ps restrained molecular dynamics (RMD) simulations. A further refinement of the DDD structure with the lowest distance errors was done by energy minimization, a prolonged RMD simulation in VUL'UO and a time-averaged RMD simulation. An average structure was obtained from a trajectory generated during 20-ps RMD. The final structure was compared with the des-(B26 -B30)-insulin crystal structure refined by molecular dynamics and the 2-Zn crystal structure of porcine insulin. This comparison shows that the overall structure of des-(B26 -B30)-insulin is retained in solution with respect to the crystal structures with a high flexibility at the N-terminal part of the A chain and at the N-terminal and C-terminal parts of the B chain. In the RMD run a high mobility of Gly A l , Asn A21 and of the side chain of Phe B25 is noticed. One of the conformations adopted by des-(B26-B30)-insulin in solution is similar to that of molecule 1 (Chinese nomenclature) in the crystal structure of porcine insulin.Due to its biological importance as a hormone and drug, insulin has been the subject of numerous studies regarding its structure and function [l]. Several crystal structures of insulins have been reported [2 -61 and a detailed picture of the spatial structure of insulins has emerged.The insulin molecule consists of two polypeptide chains, A and B, of 21 and 30 residues respectively (cf. Fig. 1). The horse-shoe-shaped A chain contains two helices (residues A2-A8 and A13-A20) and the B chain one helix (residues B9 -BI 9). These chains are connected by two disulphide bridges (A7 -B7 and A20 -B19) which, together with a third one (A6 -A1 I), help stabilize the folded form of insulin over a broad range of temperatures and pH values. In the crystal structures the last six residues of the B chain form an antiparallel fl-sheet with another insulin molecule. Insulin is a rather flexible molecule, as evidenced by large structural differences found in different crystal structures. The major differences between various forms of insulin are in the N-terminal of the B chain. For instance, the porcine 4-Zn insulin structure has an additional helical region running from B1 to B8 [4], whereas in 2-Zn insulin this fragment is in a random coil conformation. In fact, a variety of solution and crystal studies showed that the conversion of the 2-Zn to the 4-Zn structure can be induced by high concentrations of anions [7]. Adding Correspondence to R . Boelens, Department of Chemistry, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The NetherlandsAhbveviutions. Des-(3326 -B30)-insulin, insulin lacking the C-te...

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Phenol stabilizes more helix in a new symmetrical zinc insulin hexamer

Cited by 338 publications

References 12 publications

Operator-assisted harvesting of protein crystals using a universal micromanipulation robot

Operator-assisted harvesting of protein crystals using a universal micromanipulation robot

Insulin analog with additional disulfide bond has increased stability and preserved activity

The solution structure of a monomeric insulin

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