Niels C Kaarsholm

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Insulin circulates in the bloodstream and binds to its specific cell-surface receptor as a 5808 Da monomeric species. However, studies of the monomer structure and dynamics in solution are severely limited by insulin self-association into dimers and higher oligomers. In the present work we use site-directed mutagenesis of the dimer- and hexamer-forming(More)
To evaluate the possible relationship between biological activity and structural stability in selected regions of the insulin molecule, we have analyzed the guanidine hydrochloride induced reversible unfolding of a series of mutant insulins using a combination of near- and far-UV circular dichroism (CD). The unfolding curves are reasonably described on the(More)
Studies of naturally occurring and chemically modified insulins indicate that relatively few of the 51 amino acid residues may be assigned specific roles in insulin-receptor interactions. Most of the insulin X-ray structural information is derived from aggregated species (notably hexamers). Because insulin exerts its physiological effect as a 5808 Dalton(More)
The insulin hexamer is an allosteric protein capable of undergoing transitions between three conformational states: T6, T3R3, and R6. These transitions are mediated by the binding of phenolic compounds to the R-state subunits, which provide positive homotropic effects, and by the coordination of anions to the bound metal ions, which act as heterotropic(More)
One- and two-dimensional 1H NMR spectroscopy have been employed to probe the association and subsequent conformational changes of metal-free insulin in sodium chloride solution at pH 9 and 9.4. These studies establish that the proton resonances of His(B5) and His(B10) are useful signatures of aggregation and conformation. Changes in chemical shifts and(More)
Adaptation to efficient heterologous expression is a prerequisite for recombinant proteins to fulfill their clinical and biotechnological potential. We describe a rational strategy to optimize the secretion efficiency in yeast of an insulin precursor by structure-based engineering of the folding stability. The yield of a fast-acting insulin analogue(More)
Hexameric insulin is an allosteric protein that undergoes transitions between three conformational states (T(6), T(3)R(3), and R(6)). These allosteric states are stabilized by the binding of ligands to the phenolic pockets and by the coordination of anions to the His B10 metal sites. Raman difference (RD) spectroscopy is utilized to examine the binding of(More)
Site-directed mutagenesis is used in conjunction with 1H nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy in order to find an insulin species amenable for structure determination in aqueous solution by NMR spectroscopy. A successful candidate in this respect, i.e., B16 Tyr-->His mutant insulin, is identified and selected for(More)
The aggregation properties of zinc-free insulin have been studied using static and dynamic light scattering. The aggregation has been investigated as a function of three parameters, the concentration of sodium chloride (in the range 10-100 mM), the pH value (in the range pH 7.5-10.5), and the insulin concentration (1.8-13.4 mg/mL). The measured homodyne(More)
C.d. and fluorescence spectroscopy have been used to investigate the effect of ligand binding on the structure and stability of folate-binding protein (FBP) from cow's whey. The c.d. spectrum of unligated FBP predicts the following secondary structure: 22% helix, 25% antiparallel beta-strand, 5% parallel beta-strand, 17% turn and 31% random-coil structure.(More)