Probing the Impact of Temperature and Substrates on the Conformational Dynamics of the Neurotransmitter:Sodium symporter LeuT

•HDX-MS reveals the conformational impact of two substrates and temperature on LeuT.•Binding of Ala versus Leu induces distinct differences in the dynamics of LeuT.•Multiple regions in LeuT exhibited increased dynamics at elevated temperatures.•EX1 kinetics is detected at elevated temperatures and in the presence of Ala/Leu.•Both substrates and temperature modulate the slow dynamics (EX1) in LeuT.