Rational mutation of proteins based on their structural and dynamic characteristics is a useful strategy for amplifying specific fluctuations in proteins. Here, we show the effects of mutation on the conformational fluctuations and thermodynamic stability of ubiquitin. In particular, we focus on the salt bridge between K11 and E34 and the hydrogen bond between I36 and Q41, which are predicted to control the fluctuation between the basic folded state, N1
, and the alternatively folded state, N2
, of the protein, using high-pressure NMR spectroscopy. The E34A mutation, which disrupts the salt bridge, did not alter picosecond–to–nanosecond, microsecond–to–millisecond dynamic motions, and stability of the protein, while the Q41N mutation, which destabilizes the hydrogen bond, specifically amplified the N1
conformational fluctuation and decreased stability. Based on the observed thermodynamic stabilities of the various conformational states, we showed that in the Q41N mutant, the N1
state is more significantly destabilized than the N2
state, resulting in an increase in the relative population of N2
. Identifying the interactions controlling specific motions of a protein will facilitate molecular design to achieve functional dynamics beyond native state dynamics.
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