AUREMOL-QTA, a program package for NMR based automated recognition and characterization of local and global conformational changes in proteins induced by ligand binding as external perturbation

Among all existing techniques for protein structure determination, NMR spectroscopy has the advantage to provide the most complete characterization of molecular structures in solution. On the other hand, the low sensitivity of NMR limits the available signal-to-noise ratio. This often leads to the disappearance of cross peaks and to the misinterpretations of noise peaks as peaks originating from the protein under consideration. These properties hamper the determination of the consistency between the two investigated spectra. In this work, a completely new quality control (AUREMOL-QTA) package has been developed in order to automatically infer structural conformational changes from spectral modifications (in a set of investigated spectra). These differences may be induced by alterations of the external conditions (e.g. temperature, pressure, pH and ligand binding) as well as (partial) protein denaturation. The complex task of manually collecting and interpreting vicinity relationships changes (through space and bonds) in order to extract structural modifications of the molecule is time demanding. The developed AUREMOL-QTA package facilitates this investigation by means of multi-dimensional NMR spectra and it is applicable to three main possible sceneries: 1. Quality control of a protein sample and assessment of the intact three- dimensional structure of the protein. 2. Automated identification of ligand binding sites by means of NMR spectroscopy. 3. Identification of structural changes (in the atomic resolution) induced by external perturbations such as pressure, pH and temperature. The AURMOL-QTA begins with a pre-processing step where one (or more) reference spectra of the target protein and one or more test spectra are normalized (spectral width, offset, receiver gain and the number of scans) to be properly compared. A possible spectrum shift is evaluated and if it has occurred it is corrected. The simulated spectrum of a protein can be used as an adjunctive reference spectrum and to recognize peak multiplets in the experimental spectra. If there are more reference spectra only the common peaks are retained and used for the requested comparison. The routine continues with a low-level analysis involving a peak feature collection (volume, line width, chemical shift, cross-correlation in the time domain and shape) with a consequent association of cross peaks among the spectra. A mid-level analysis relies on the calculation of feature-related Bayesian probabilities that the associated peaks represent the same signal. It facilitates the detection of missing signals and the identification of feature differences between the compared spectra. The ratio of matching peaks is computed in order to quantitatively determine the similarity of the investigated spectra. A high-level analysis allows the identification of structurally altered parts of the molecule, mapping the feature variations and computing the fraction of residues involved in the modifications. In particular, if NOESY and TOCSY spectra are investigated the symmetrical properties are exploited in order to perform an adjunctive pattern analysis of the residues. The method has been successfully tested on HSCQ spectra (pressurized with xenon) and on HSQC-TROSY spectra (with high pressure and temperature variations) of the human prion protein (huPrPC). It has been also used to compare the NOESY spectrum of the wild HPr protein from Staphylococcus aureus with the mutant (H15A) of the same protein and with the artificially denatured (partially and totally) ones.