Probing protein structure by solvent perturbation of NMR spectra: the surface accessibility of bovine pancreatic trypsin inhibitor

Abstract

In the absence of specific interactions, the relative attenuation of protein NMR signals due to added stable free radicals such as TEMPOL should reflect the solvent accessibility of the molecular surface. The quantitative correlation between observed attenuation and surface accessibility was investigated with a model system, i.e., the small protein bovine pancreatic trypsin inhibitor. A detailed discussion is presented on the reliability and limits of the approach, and guidelines are provided for data acquisition, treatment, and interpretation. The NMR-derived accessibilities are compared with those obtained from x-ray diffraction and molecular dynamics data. Although the time-averaged accessibilities from molecular dynamics are ideally suited to fit the NMR data, better agreement was observed between the paramagnetic attenuations of the fingerprint cross-peaks of homonuclear proton spectra and the total NH and H alpha accessibilities calculated from x-ray coordinates, than from time-averaged molecular dynamics simulations. In addition, the solvent perturbation response appears to be a promising approach for detecting the thermal conformational evolution of secondary structure elements in proteins.

Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry

“…The ␤2m mutant and the stock ␣B-crystallin solutions employed for measuring the signal attenuations were filtered using 0.02-m microfilters with either lyophilized or un-lyophilized purified ␤2m proteins being used. The 1 H two-dimensional total correlation spectroscopy (TOCSY) (52) NMR spectra were collected and processed as reported previously (19,53). Based on the signal-to-noise ratio, amplitude errors typically ranged within Ϯ5%, although much larger errors are to be expected for very weak resonances.…”

NMR Studies of Protein Surface Accessibility

“…Water resonance was attenuated using a DANTE presaturation train superimposed on the specific sequence. Additional experimental parameters were the same as those described previously (7,17). A total of 470 increments were collected in t 1 , with 1908 data points and 64 scans/FID in t 2 , over a spectral width of 6 kHz in both dimensions.…”

“…Water suppression was achieved through an excitation sculpting (29) module appended to the sequences (13,14). The experimental conditions of TOCSY, nuclear Overhauser enhancement and exchange spectroscopy, and 1 H-13 C heteronuclear single quantum coherence spectra have been described elsewhere (7,17). The decrease of peak intensities caused by the added TEMPOL was evaluated from a comparison of ePHOGSY-TOCSY with NOE spectra obtained in the presence and absence of the paramagnetic probe and performed and processed with the same parameters.…”

“…A combined use of paramagnetic perturbation and water-protein Overhauser spectroscopy based on 1D 1 and 2D ePHOGSY (13,14) can prove this link and may thus yield a faithful description of the static and dynamic properties of the water blanket. The attenuation of a specific set of hydrogen resonances in the NMR spectrum in the presence of a soluble paramagnetic probe reflects the accessibility of the corresponding locations on the protein surface (17). On the other hand, ePHOGSY experiments trace out different types of water-protein interactions by means of an initial intermolecular magnetization transfer step due to chemical exchange or dipolar interactions or both processes.…”

Rapid Mapping of Active Site of KSI by Paramagnetic NMR

“…[25][26][27] Line-broadening of NMR resonances may relate to solvent exposure of residues in the protein. KSI has a hydrophobic cavity with approximate dimensions of 8.5 × 9.5 Å surface and 16 Å deep.…”