G-protein βγ (Gβγ) subunits interact with a wide range of molecular partners including: Gα subunits, effectors, peptides, and small molecule inhibitors. The molecular mechanisms underlying the ability to accommodate this wide range of structurally distinct binding partners are not well understood. To uncover the role of protein flexibility and alterations in protein conformation in molecular recognition by Gβγ, a method for site-specific ¹⁵N-labeling of Gβ-Trp residue backbone and indole amines in insect cells was developed. Transverse Relaxation Optimized Spectroscopy-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance (TROSY-HSQC NMR) analysis of ¹⁵N-Trp Gβγ identified well-dispersed signals for the individual Trp residue side chain and amide positions. Surprisingly, a wide range of signal intensities was observed in the spectrum, likely representing a range of backbone and side chain mobilities. The signal for GβW99 indole was very intense, suggesting a high level of mobility on the protein surface and molecular dynamics simulations indicate that GβW99 is highly mobile on the nanosecond timescale in comparison with other Gβ tryptophans. Binding of peptides and phosducin dramatically altered the mobility of GβW99 and GβW332 in the binding site and the chemical shifts at sites distant from the direct binding surface in distinct ways. In contrast, binding of Gα_i1-GDP to Gβγ had relatively little effect on the spectrum and, most surprisingly, did not significantly alter Trp mobility at the subunit interface. This suggests the inactive heterotrimer in solution adopts a conformation with an open subunit interface a large percentage of the time. Overall, these data show that Gβγ subunits explore a range of conformations that can be exploited during molecular recognition by diverse binding partners.