The complex of spliceosomal U1A protein and its cognate SL2 RNA is a prototype system for protein-RNA binding studies. A major question is whether U1A protein alone is capable of undergoing conformational dynamics similar to structural rearrangements upon RNA binding. Using a fast temperature jump and tryptophan fluorescence detection, we uncover a similar to 20 mu s conformational transition for the Lys22Gln/Phe56Trp-only mutant of U1A, yet a Phe56Trp-only control mutant does not show the transition. To explain this observation, we performed extensive molecular dynamics (MD) simulations. The simulations explain why only the Lys22Gln/Phe56Trp-only mutant shows a fluorescence signal: in the other mutant, the tryptophan probe is not quenched upon structural rearrangement. The simulations support helix C movement as the underlying structural rearrangement, although the simulated time scale is faster than experimentally detected. On the basis of our MD results, we propose a reversible two-pathway three-state transition for the helix C movement and assign T-jump kinetics to a closed to semi-closed transition of the helix. Our result provides a specific example of how alternative protein conformations on the native side of the folding barrier can be functionally important, for example in conformational selection by a binding partner.