Mutagenic studies that implicate specific amino acids in reaction chemistry or in substrate binding are fully and accurately interpretable only when the structural integrity of the variant protein has been validated. This validation is facilitated by identification of spur-labeled metabolic analogs. Measurement of spin-probe affinity and binding stoichiometry, using K13M mutant and comparison of these parameters with the values for wild-type mevalonate kinase affords an elegant, rigorous, yet efficient solution-state method for validating the structural integrity of the engineered mutant enzyme. The spin-probe ATPSAP is being productively used to study the active site structure of mevalonate kinase which catalyzes a key step in cholesterol biosynthesis. Studies were conducted using X-band EPR spectroscopy.