Ras homology protein (RhoA), a small G protein (SmG), plays a crucial pathophysiological role and is implicated in cardiovascular diseases such as diabetes, heart failure, hypertension, and stroke. RhoA regulates cytoskeleton remodeling through actin fibrillation and aids in overall cell survival. Like all SmGs that function as regulatory switches, the amount of RhoA in the heart is delicately controlled, with large changes resulting in faulty signaling that can lead to cardiac disease. ∼2-fold overexpression protects myocytes from ischemic injury and decreases fibrosis during the stress response, while a large excess (∼20-fold) is associated with cardiac hypertrophy, fibrosis, and cardiomyopathy. A myriad of studies have been conducted on the structure-function relationship of SmGs, with most structural biology and biophysical investigations employing a reductionist in vitro strategy in which the native physiological context is ignored. In order to assess how proteins work in vivo, there is a growing emphasis to investigate systems in a native physiological environment. In this presentation, I will report on my investigation of conformational dynamics and enzymatic activity of the RhoA protein in a crowded environment. I will discuss RhoA behavior using 2D 15N-1H NMR, 19F NMR and fluorescence spectroscopy and will present my findings on both the enzyme activity and conformations of GDP- and GTP- bound RhoA under the influence of macromolecular crowders, small molecule metabolite crowding, and the role of cardio-specific metabolites. In particular, I will use 19F nuclei as robust cellular NMR tracers and reporters on live in-cell using19F labeled RhoA protein