Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra

Abstract

The effects induced by extrinsic paramagnetic probes on protein NMR spectra, widely used for surface mapping, can also be exploited to detect the sites of slow and intermediate exchange due to structural or intermolecular interaction dynamics.

Insights into a Protein-Nanoparticle System by Paramagnetic Perturbation NMR Spectroscopy

“…Using extrinsic spin labels such as nitroxides to extract structural information requires testing the reliability of their non-specific probe behavior [17][18][19][20]. ESR spectra of Tempol in the absence and presence of β2m had previously shown that only statistical encounters occur between the free radical and the protein, as inferred from the invariance of linewidths and amplitudes of the superimposed spectra [16]. For the ternary system protein + AuNPs + Tempol, the ESR trace superposition for the three controls (Tempol, Tempol + AuNPs, and Tempol + β2m) and the ternary system shows substantial coincidence with some small amplitude deviations ( Figure S1).…”

“…Therefore, the occurrence of Tempol interactions other than the statistical collision in the binary and ternary systems here considered should be ruled out. [16][17][18], A N [eq] values larger or smaller than unity indicate amide signals attenuated above or below the average attenuation, respectively, and therefore, they identify molecular locations more or less accessible to the nitroxide probe, depending on the specific surface exposure. Instead, the interpretation of the A N [off-eq] values is related to their relationship with the corresponding A N [eq] figures [16].…”

“…Recently, we have revived the use of water-soluble nitroxides such as Tempol to explore the exchange dynamics of β2m [16]. Tempol and similar stable free radicals had been formerly employed as extrinsic probes for identifying the protein exposed locations, based on the paramagnetic perturbation of the NMR signals induced by the unpaired electron of the radical over accessible molecular surfaces [17][18][19][20].…”

“…The same paramagnetic perturbation measured under non-equilibrium conditions of the NMR magnetization determines an attenuation pattern that differs from the corresponding profile obtained under equilibrium conditions, i.e., with fully relaxed NMR magnetization. While in the latter conditions, the extent of NMR attenuation reflects the proximity to the unpaired electron and therefore the accessibility of or the distance from the molecular surface, the attenuation retrieved under non-equilibrium conditions of magnetization recovery can map also the locations of hindered accessibility or µs-to-ms exchange events, by identifying slower or faster relaxing nuclei, respectively, with respect to the average relaxation rate enhancement brought about by the nitroxide probe [16].…”

“…Here, we show the application of this novel use of Tempol attenuation to gain insights into the interactions that wild-type β2m establishes with citrate-coated AuNPs. The analysis of the ternary system protein/AuNP/spin-label probe is conducted with respect to all the NMR-(Nuclear Magnetic Resonance) and ESR-(Electron Spin Resonance) accessible controls involving only two components of the system, which are based also on the previously reported evidence [2][3][4][5][6][7]16].…”