Double electron-electron resonance (DEER) has been used to show that sequence variations in HIV-1 protease (HIV-1PR) subtypes result in different average flap conformational ensembles. Inhibitor binding is shown to induces shifts in flap conformational sampling. 15N-HSQC titration methods are used to estimate inhibitor binding strength by observing changes in the NMR peak patterns. Because DEER data are collected at 65K, here, we use solution NMR to confirm DEER results. The HSQC titration results support the results of DEER and suggest that it can be used to study highly dynamic enzyme system such as HIV-1PR upon interaction with inhibitors and substrates. In the future, 15N CMPG dispersion experiments will be performed to analyze the exchange rate and population of flap conformational states, which help to provide direct robust support to DEER methodology.

Measurement of flap conformational sampling and binding interaction changes of HIV-1 protease variants

244th National Meeting (2012)

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