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Mutations associated with disease states or developmental abnormalities can be exploited as “nature-guided” probes of structure and function. These studies are invaluable to uncovering molecular mechanisms of function for a given protein or gene. Especially when mutations are inherited or exhibit haploinsuffiency phenotypes for paired gene alleles. We have exploited one such library of mutations located within the DNA binding domain of a transcription factor that initiates male sex determination, SRY (sex determining region of the Y chromosome). DNA binding of SRY is mediated through the HMG (high mobility group) box, a small DNA binding and bending domain conserved among an entire transcription factor family. Clinical mutations in the HMG box of human SRY are present throughout the entirety of the domain. Additionally, these mutations represent different genetic backgrounds and can be inherited from a normally developed father. Coupled to the high degree of conservation in amino acid composition and tertiary structure, the use of these “experiments of nature” elucidate molecular mechanisms of human sex reversal and provide molecular insight into an entire architecture transcription factor family. Within the following three chapters the clinical mutations associated with disorders of sexual development serve as probes of structure and function for a prototypical domain for a family of architectural transcription factors. Initially we exploit a high-throughput assay to determine retained high-affinity specific DNA binding for the entire set of missense mutations (Chapter 1). To the best of our knowledge a side-by-side characterization of all the clinical mutations the HMG box has not been described. Subsequent chapters (Chapter 3 and 4) utilize clinical mutations as structure-function probes. A cavity-creating de novo mutation in the hydrophobic core of the HMG box leads to a “perfect storm” of defects including insignificant transcriptional activation of the full length human SRY (Chapter 3). This is in stark contrast to characterization of an inherited mutation also in the core of the HMG box domain (Chapter 4). The inherited mutation exhibits a twofold reduction in the transcriptional activation of the male-specific genetic regulatory network; suggesting that SRY functions just above a critical threshold required for proper pathway activation.