Protein resonance

The intense water signal and the broad protein resonances were suppressed by a combination of continuous secondary Irradiation at the water frequency and the Hahn spin-echo sequence (0[90 x-t-180 y-t-collect]). 

Other enzyme-substrate or inhibitor interaction studies80 82 have been addressed, using a combination of STD and trNOE NMR experiments, in order to collect details on the substrate bound conformation (ligand perspective). In other cases, the availability of a labelled protein receptor83 have permitted to follow the induced chemical shift variations of the protein resonances upon ligand addition to the NMR tube by HSQC methods (protein perspective). 

Eq. (15) and the following discussion are couched in terms of ligand resonances, but exactly the same considerations apply to protein resonances. 

X-ray crystallography, docking modes can be validated by various NMR techniques NOEs may be observed between the ligand and the receptor protein by heteronuclear-filtered NOE spectroscopy [51], chemical shift changes of protein resonances upon binding can be analyzed by simulation of shifts caused by ring currents and electrostatic effects [52], and saturation transfer difference measurements indicate which part of the ligand is in direct contact with the protein [52]. 

Finally, a saturation-transfer screening (using short saturation times) of the protein resonance envelope to identify irradiation frequencies that yield large STDs may be of some aid in the identification of residues within the binding pocket, and in assignments for the calculations. 

Han S, Czernuszewicz RS, Kimura T, et al. 1989a. Fe2S2 protein resonance Raman revisited structural variations among adrenodoxin, ferredoxin, and red paramagnetic protein. J Am Chem Soc 111 3505-11. 

Figure 5,20 Portion of a 3D X-filtered NOESY spectrum of uniformly 13C/15N-labeled, stability-enhanced kinaseX in complex with kinaseX inhibitor 2. The protein and inhibitor concentrations used were 300 pM. The F3 (inhibitor1H) plane is at 7.83 ppm. Peaks with protein resonance assignments are labeled. (Note Val-A and Val-B refer to the y-i and y methyl, respectively of the same valine residue.) The spectrum was recorded at 35 °C, 600 MHz 1H frequency using a NOESY mixing time of 100 ms on a Varian Inova spectrometer equipped with a Cold Probe. The spectrum is aliased in the 13C (F2) dimension.

Saturation transfer difference NMR stands for a difference experiment in which the protein resonances are selectively saturated but not the resonances of the ligand [38], In the second experiment, the proton resonances of neither the ligand nor the protein are saturated. If the ligand binds to the protein with a Kd between mM and... 

Fig. 38 STD NMR spectra map the close contacts of I19L with tubulin, a One-dimensional lH NMR spectrum of I19L in the presence of tubulin. b,c One-dimensional STD-NMR spectra of I19L in the presence of tubulin with selective saturation of protein resonances at 0 and 10 ppm, respectively. Protons of I19L affected by the selective saturation of tubulin are labeled. (Reprinted with permission from [142]. Copyright 2005 American Chemical Society)...

These data can be interpreted as follows. Since the chemical shifts of both resonances from the protein and the inhibitor are concentration dependent, the exchange rate between the free and bound states is fast on the NMR time scale. The observed chemical shifts are a weighted average of the two states [Eq. (10.13)]. For the protein resonances, the fraction bound increases with increasing inhibitor concentration. At zero concentration of the inhibitor, the observed chemical shift is that of the unbound state, while at concentrations above -0.06 M inhibitor, the observed chemical shift is that of the bound state. For the inhibitor, the highest fraction bound occurs at the lower concentrations of the inhibitor and the lowest fraction bound at the higher concentrations. There-... 

Figure 7 Illustration of the use of saturation transfer to assign H NMR resonances of one form of a metalloprotein based on known assignments of another form of the protein. Here, heme methyl H NMR resonances of a folding intermediate of horse ferricytochrome c in exchange with the native form are assigned, (a) Reference 500-MHz H NMR spectrum, 90% H2O 10% D2O, 55 °C, 5.4 M urea, (b, c) Difference spectra showing saturation transfer observed (90% H20 10% D2O, 55 °C, 5.4 M urea) upon irradiating the native protein resonances at (b) 32.1 ppm (native 8-CH3) and (c) 29.8 ppm (native 3-CH3), allowing assignment of L and in species L to heme 8-CH3 and 3-CH3, respectively. (Adapted from Russell, Melenkivitz and Bren )...

A second variation of saturation transfer experiment has been devised by Dalvit and coworkers that uses the transfer of magnetization from the water (167). Water is intimately associated with proteins being bound either within or on the surface of the macromolecu-lar structure. Saturation of the water resonance will lead to protein saturation through a variety of mechanisms, including saturation of the aH resonances, saturation of exchanging protein resonances, and NOE interactions between water and the protein. If a compound is bound to the protein it will also become saturated, and this effect can be used as an indication of ligand binding (167). 

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