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Mammalian testis-determining factor SRY contains a high mobility group box, a conserved eukaryotic motif of DNA bending. Mutations in SRY cause XY gonadal dysgenesis and somatic sex reversal. Although such mutations usually arise de novo in spermatogenesis, some are inherited and so specify male development in one genetic background (the father) but not another (the daughter). Here, we describe the biophysical properties of a representative inherited mutation, V60L, within the minor wing of the L-shaped domain (box position 5). Although the stability and DNA binding properties of the mutant domain are similar to those of wild type, studies of SRY-induced DNA bending by subnanosecond time-resolved fluorescence resonance energy transfer (FRET) revealed enhanced conformational fluctuations leading to long range variation in bend angle. 1H NMR studies of the variant protein-DNA complex demonstrated only local perturbations near the mutation site. Because the minor wing of SRY folds on DNA binding, the inherited mutation presumably hinders induced fit. Stopped-flow FRET studies indicated that such frustrated packing leads to accelerated dissociation of the bent complex. Studies of SRY-directed transcriptional regulation in an embryonic gonadal cell line demonstrated partial activation of downstream target Sox9. Our results have demonstrated a nonlocal coupling between DNA-directed protein folding and protein-directed DNA bending. Perturbation of this coupling is associated with a genetic switch poised at the threshold of activity. [ABSTRACT FROM AUTHOR]

Insulin is susceptible to fibrillation, a misfolding process leading to well ordered cross-β assembly. Protection from fibrillation in β cells is provided by sequestration of the susceptible monomer within zinc hexamers. We demonstrate that proinsulin is refractory to fibrillation under conditions that promote the rapid fibrillation of zinc-free insulin. Proinsulin fibrils, as probed by Raman microscopy, are nonetheless similar in structure to insulin fibrils. The connecting peptide, although not well ordered in native proinsulin, participates in a fibril-specific β-sheet. Native insulin and proinsulin exhibit similar free energies of unfolding as inferred from guanidine denaturation studies: relative amyloidogenicities are thus not correlated with global stability. Strikingly, the susceptibility of proinsulin to fibrillation is increased by scission of the connecting peptide at single sites. We thus propose that the connecting peptide constrains a large scale conformational change in the misfolded protein. A tethering mechanism is proposed based on a model of an insulin protofilament derived from electron-microscopic image reconstruction. The proposed relationship between cross-β assembly and protein topology is supported by studies of single-chain analogs (mini-proinsulin and insulin-like growth factor I) in which foreshortened connecting peptides further retard fibrillation. In addition to its classic function to facilitate disulfide pairing, the connecting peptide may protect β cells from toxic protein misfolding in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Insulin fibrillation provides a model for a broad class of amyloidogenic diseases. Conformational distortion of the native monomer leads to aggregation-coupled misfolding. Whereas β-cells are protected from proteotoxicity by hexamer assembly, fibrillation limits the storage and use of insulin at elevated temperatures. Here, we have investigated conformational distortions of an engineered insulin monomer in relation to the structure of an insulin fibril. Anomalous 13C NMR chemical shifts and rapid 15N-detected ¹H-²H amide-proton exchange were observed in one of the three classical a-helices (residues A1-A8) of the hormone, suggesting a conformational equilibrium between locally folded and unfolded A-chain segments. Whereas hexamer assembly resolves these anomalies in accordance with its protective role, solid-state 13C NMR studies suggest that the A-chain segment participates in a fibril-specific β-sheet. Accordingly, we investigated whether helicogenic substitutions in the A1-A8 segment might delay fibrillation. Simultaneous substitution of three E-branched residues (IleA2 → Leu, ValA3 → Leu, and ThrA8 → His) yielded an analog with reduced thermodynamic stability but marked resistance to fibrillation. Whereas amide-proton exchange in the A1-A8 segment remained rapid, 13Cα chemical shifts exhibited a more helical pattern. This analog is essentially without activity, however, as IleA2 and ValA3 define conserved receptor contacts. To obtain active analogs, substitutions were restricted to A8. These analogs exhibit high receptor-binding affinity; representative potency in a rodent model of diabetes mellitus was similar to wild-type insulin. Although 13Cα chemical shifts remain anomalous, significant protection from fibrillation is retained. Together, our studies define an "Achilles' heel" in a globular protein whose repair may enhance the stability of pharmaceutical formulations and broaden their therapeutic deployment in the developing world. [ABSTRACT FROM AUTHOR]