Protein Folding Monitored at Individual Residues During a Two-Dimensional NMR Experiment

Abstract

An approach is described to monitor directly at the level of individual residues the formation of structure during protein folding. A two-dimensional heteronuclear nuclear magnetic resonance (NMR) spectrum was recorded after the rapid initiation of the refolding of a protein labeled with nitrogen-15. The intensities and line shapes of the cross peaks in the spectrum reflected the kinetic time course of the folding events that occurred during the spectral accumulation. The method was used to demonstrate the cooperative nature of the acquisition of the native main chain fold of apo bovine alpha-lactalbumin. The general approach, however, should be applicable to the investigation of a wide range of chemical reactions.

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

“…All NMR signals observed during the folding process can be assigned either to the MG or the native state. No additional peaks indicative of a significantly populated folding intermediate were detected, in agreement with previous findings (23,26,27). The refolding kinetics could be quantified for 92 of 121 backbone amide sites in the protein from intensity measurements of well resolved cross-peaks in the 1 H-15 N correlation spectra (Fig.…”

“…2b). In addition, and in contrast to the studies by Balbach et al (23,26,27), the intensity decay of five cross-peaks characteristic for the MG state could be quantified. Under the experimental conditions chosen (15°C, pH 8, Ca 2ϩ -free), the NMR intensity decay and buildup curves can be fitted to monoexponential functions.…”

“…Additional diffusion-edited and NOE transfer NMR measurements allowed monitoring the compactness of the protein and the establishing of native tertiary contacts, respectively (23,27). The folding kinetics of apo-␣-lactalbumin have also been studied previously at a residue level by line shape analysis of individual cross-peaks detected in a single 2D NMR spectrum recorded during the refolding event (26). This technique, however, is limited to relatively slow kinetics (several minutes) and is prone to large experimental uncertainties.…”

“…This technique, however, is limited to relatively slow kinetics (several minutes) and is prone to large experimental uncertainties. In the study of Balbach et al (26), folding rates for a total of 25 backbone amides could be quantified with an estimated experimental error of Ϸ25%. Because of the small number and the high uncertainties of the measured rate constants, these data did not allow definite exclusion of the presence of differential folding kinetics along the polypeptide chain.…”

Time‐resolved NMR studies of RNA folding

“…The spectrum contains the resonances of the different states modulated by their respective kinetics. 104 For faster processes, recording of a sensitive experiment with high chemical shift dispersion along the indirect dimension, such as a HSQC experiment is possible 105 ; for slow processes, also the application of experiments is suitable that require longer total experimental time, such as NOESY experiments. 51 The resulting line-shapes are modulated by the result of the FT of the kinetic process along the indirect dimension.…”