The RNA recognition motif (RRM), one of the most common RNA-binding domains, recognizes sing
le-stranded RNA. A C-termina
l he
lix that undergoes conformationa
l changes upon binding is often an importantcontributor to RNA recognition. The N-termina
l RRM of the U1A protein contains a C-termina
l he
lix (he
lixC) that interacts with the RNA-binding surface of a
![](/images/gifchars/beta2.gif)
le">-sheet in the free protein (c
losed conformation), but isdirected away from this
![](/images/gifchars/beta2.gif)
le">-sheet in the comp
lex with RNA (open conformation). The dynamics of he
lix C inthe free protein have been proposed to contribute to binding affinity and specificity. We report here a directinvestigation of the dynamics of he
lix C in the free U1A protein on the nanosecond time sca
le using time-reso
lved f
luorescence anisotropy. The resu
lts indicate that he
lix C is dynamic on a 2-3 ns time sca
le withina 20
![](/images/entities/deg.gif)
range of motion. Steady-state f
luorescence experiments and mo
lecu
lar dynamics simu
lations suggestthat the dynamica
l motion of he
lix C occurs within the c
losed conformation. Mutation of a residue on the
![](/images/gifchars/beta2.gif)
le">-sheet that contacts he
lix C in the c
losed conformation dramatica
lly destabi
lizes the comp
lex (Phe56A
la)and a
lters the steady-state f
luorescence, but not the time-reso
lved f
luorescence anisotropy, of a Trp in he
lixC. Mutation of Asp90 in the hinge region between he
lix C and the remainder of the protein to A
la or G
lysubt
ly a
lters the dynamics of the U1A protein and destabi
lizes the comp
lex. Together these resu
lts show thathe
lix C maintains a dynamic c
losed conformation that is stab
le to these targeted protein modifications anddoes not equi
librate with the open conformation on the nanosecond time sca
le.