Studying IDPs using CCR experiments of high dimensionality

For many years, it was believed that every protein possesses a rigid three-dimensional structure, tightly related to its function. However, since 1980s more and more flexible proteins have been observed, that only transiently adopt certain structural motifs. Surprisingly, they were still able to fulfill physiological functions. Moreover, for some of them the structural flexibility was crucial for the functionality. These so-called intrinsically disordered proteins (IDPs) became important objects to study. Nuclear magnetic resonance (NMR) is the only experimental technique which allows to investigate structural details of IDPs. In an NMR experiment, a protein is placed in a high magnetic field and subjected to a sequence of radio-frequency electromagnetic pulses. As a result, certain nuclei are excited. Subsequently, they come back to equilibrium state, in a process of relaxation. During that process, their signal is recorded and presented in a form of spectrum multidimensional chart, in which the characteristic peaks appear at certain positions, different for various proteins. Certain NMR experiments allow to identify residual structural motifs present in an IDP under investigation, which may reflect the proteins way of operation. Cross-correlated relaxation measurement is one class of such experiment. Here, the structural information is coded in peaks intensities. The problem with NMR studies of IDPs is that the spectral peaks of these proteins are typically distributed in a very narrow range and thus we have to cope with peak overlap, which significantly complicates the analysis. The possible solution to this problem is to increase the dimensionality of the spectrum. Then, the peaks are distributed over a bigger space (e.g. five-dimensional cube instead of three-dimensional one). The aim of this project is to incorporate high-dimensional spectra into cross-correlated relaxation measurements of IDPs. To achieve this goal, we need to develop the new methods of exciting the IDPs nuclei using the magnetic field pulses, so that the spectrum would be of desired dimensionality. Different types of such experiments would provide different types of structural information, for instance various angles between the protein bonds would be measured. Such diversity would yield a consistent picture of a studied molecule. A suite of high-dimensional experiments developed during the project will make accessible structure- related data of IDPs providing especially difficult spectra with overlapping peaks. Unambiguous interpretation will make the investigation process easy, fast and reliable. The novel methods will be applied to two biologically important IDPs, BASP1 and osteopontin, playing important roles in neuroplasticity and tumor growth.

In living organisms proteins fulfill many physiological functions. Their function is often determined by their shape or three-dimensional structure. However there is a large class of proteins, called intrinsically disordered proteins that lack such a rigid structure. They only transintly adopt some structural motifs, being very mobile. The mobility does not prevent them from fulfilling their function; even more: very often this function can be fulfilled thanks to the high mobility. The aim of structural studies of IDPs is determination of a structural ensemble a set of structures that can be adopted by an IDP. The aim of the project was to develop the nuclear magnetic resonance (NMR) methods to study structural properties of intrinsically disordered proteins. NMR is the best experimental technique for studying IDPs, which allows to study proteins with atomic resolution. However IDPs are relatively difficult objects to study. Due to the high flexibility, their NMR signals feature high level of overlap. To overcome this problem, we employed four-dimensional techniques, which allow to separate the signals over a larger spectral space and therefore to acquire the needed information. The techniques that we developed, involved the effect of so-called cross-correlated relaxation (CCR). Relaxation is a process of getting to the equilibrium state by the studied object, after being excited. In NMR there are several mechanisms of relaxation. It is possible to measure the effect of interference of such different relaxation mechanisms. Interestingly, the size of such cross-correlation effect depends on the values of torsion angles between proteins bonds. However, in a case of IDPs, the situation is more complex. There is no single value of each angle the protein moves and we observe the average of angles over the structural ensemble. To get a complete picture of the studied IDP, several different cross-correaltion effects should be measured, giving different type of information on those angles. Within the project, we developed three new CCR experiments suitable for IDPs. Moreover, a few previously published experiments (used before for proteins with a rigid structure), were adapted for IDPs by extending them to four dimensions.