Differences in Conformational Dynamics of Ribonucleases A and S as Observed by Infrared Spectroscopy and Hydrogen–Deuterium Exchange

Native-state hydrogen-exchange studies of a fragment complex can provide structural information about the isolated fragments

“…The 2D NMR spectra for RNase A and RNase S were used to Although the x-ray structures as well as NMR spectra for RNase A and RNase S are similar (14,17), most protons in RNase A have protection factors 10 to 100-fold higher than in RNase S. Fig. 3 A and B show the PFs for amide protons of RNase A and RNase S. The lower protection factors in RNase S relative to RNase A have been generally ascribed to an increased dynamic flexibility of the RNase S complex relative to folded RNase A (20,21). In the present work, we show that this view is incorrect and propose an alternative model to account for the hydrogen exchange data.…”

“…An NMR structure is available (14,15) for RNase A with preliminary data for RNase S. Refined 3D x-ray structures of RNase A and RNase S are available at high resolution (16,17). The two proteins have very similar structures and enzymatic activity but exhibit significant differences in their dynamics and stability (18)(19)(20)(21). RNase S has been used as a model system to study the thermodynamics of protein folding and stability (22)(23)(24)(25).…”

“…At room temperature, RNase S is readily cleaved by trypsin, whereas RNase A is indefinitely stable (26,27). Both tritium exchange (28) and Fourier-transformed IR-deuterium exchange studies (21,29,30) have reported that rates of exchange of amide protons from RNase S are faster than those of RNase A (31). Data from thermodynamic studies have also been used to support the assertion that RNase S is more flexible than RNase A (20).…”

Protein Motions Are Coupled to the Reaction Chemistry in Coenzyme B12‐Dependent Ethanolamine Ammonia Lyase

“…There are also differences around 1425-1475 cm À1 , which coincide with the amide II bands of deuterated proteins. [29] The lower concentration of the protein sample (about 200 mm) compared to the free cofactor (about 8 mm) meant that we had to average more spectra to acquire good quality data, and therefore collected fewer data points. Consequently, the principle kinetics from single value decomposition analysis only fit to the sum of two exponentials as opposed to the four exponential fit to the AdoCbl data (see Figure S1 and Table S1 in the Supporting Information).…”

Water Penetration into Protein Secondary Structure Revealed by Hydrogen−Deuterium Exchange Two-Dimensional Infrared Spectroscopy

“…5 IR is an appealing technique for kinetic studies, since it can be used in combination with fast unfolding experiments. 6,7 However, AmII is not otherwise structurally sensitive and is typically overlapped with side-chain absorptions. Using twodimensional infrared (2D IR) spectroscopy, we have performed HX experiments that combine the solvent-exposure sensitivity of AmII with the secondary-structure sensitivity of amide I (AmI).…”