Ovarian suppression via Gonadotropin releasing hormone analog (GnRHa) treatment should remain as an unproven method of fertility preservation due to lack of biological plausibility, conflicting results, and design flaws in the studies which include the lack of placebo and blinding and the utility of improper surrogates for fertility and ovarian reserve.

Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of trimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0M TMAO increased the free energy change (ΔG(0)) significantly from 2.1 to 3.8kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins.

Trp fluorescent spectra appear as a log-normal function but are usually analyzed with λmax, full width at half maximum, and the first moment of incomplete spectra. Log-normal analyses have successfully separated fluorescence contributions from some multi-Trp proteins but deviations were observed in single Trp proteins. The possibility that disparate rotamer environments might account for these deviations was explored by moment spectral analysis of single Trp mutants spanning the sequence of tear lipocalin as a model. The analysis required full width Trp spectra. Composite spectra were constructed using log-normal analysis to derive the inaccessible blue edge, and the experimentally obtained spectra for the remainder. First moments of the composite spectra reflected the site-resolved secondary structure. Second moments were most sensitive for spectral deviations. A novel parameter, derived from the difference of the second moments of composite and simulated log-normal spectra correlated with known multiple heterogeneous rotamer conformations. Buried and restricted side chains showed the most heterogeneity. Analyses applied to other proteins further validated the method. The rotamer heterogeneity values could be rationalized by known conformational properties of Trp residues and the distribution of nearby charged groups according to the internal Stark effect. Spectral heterogeneity fits the rotamer model but does not preclude other contributing factors. Spectral moment analysis of full width Trp emission spectra is accessible to most laboratories. The calculations are informative of protein structure and can be adapted to study dynamic processes.