The PHOGSY (Protein Hydration Observed by Gradient SpectroscoY) experiment have been designed for the observation of protein hydration and measurement of the exchange lifetime of the amide protons and of the bulk water-bound water.

Implementation on any AVANCE spectrometer equipped with pulsed field gradients (PFGs) and inverse probehead.

The basic pulse sequence was published in 95JB306 . Improved versions have been proposed ( 95JMRB334-109 , 96JMRB282-112 , and 98JMR70-135 ) in which water selective excitation is achieved by a SPFGE scheme. It has the advantage that there is no radiation damping. After this, a NOE- or a ROE-step is inserted in which water magnetization is partially transferred via chemical exchange or via dipolar cross relaxation to some amide protons of the protein. The magnetization of these spins is then labeled in F1 with the 15N frequency via an HSQC building block including watergate suppression. PFG are including to simply destroy unwanted magnetization and some phase cycling is needed to assure selection of the desired magnetization. These sequences would be equivalent to a water-selective NOESY-HSQC and ROESY-HSQC experiments. In addition, homonuclear NOESY and TOCSY steps (water-selective NOESY-HSQC-NOESY and NOESY-HSQC-TOCSY experiments)can be appended to the PHOGSY experiment in order to relay the magnetization to other spins and, hence, facilitate the assignments of the residues containing fast-exchanging amnide resonances. In this cases, watergate can not be applied and, therefore, coherence selection is achieved with the P/N approach.

Recently, a related gradient-enhanced pulse sequence based on heteronuclear cross-polarization (Exchange-edited HEHAHA experiment) has been proposed to improve the sensitivity of fast exchanging protons ( 98JMR70-135 ).

The ge-2D inverse J-resolved experiment can be recorded in routine/automation modes and minor changes are required if a predefined parameter set is available. Parameters for water selective excitation and mixing periods must be specified.

Tutorials: 2D inverse experiments

Tutorials: 2D gradient-based inverse experiments

The resulting 2D spectrum shows the typical heteronuclear 1H-X correlations. The intensities of cross-peaks give information about the solute-water exchange.

Related experiments:

2D Inverse experiments

2D Inverse gradient-enhanced experiments