Project: „Novel ¹³C-detected NMR techniques

for resonance assignment of intrinsically disordered proteins”

Project coordinator: Dr Anna Zawadzka-Kazimierczuk

Project realization: March 2013 - June 2015

The project is realized within the PARENT-BRIDGE programme ("POMOST")

of Foundation for Polish Science, cofinanced from European Union, Regional Development Fund.

 

 

Description of the project

Proteins, being involved in life processes of all living organisms, constitute very important objects for structural studies. For many years, the structure-function paradigm, associating protein’s function with its three-dimensional structure, was a central assumption of molecular biology. Indeed, under physiological conditions, proteins usually adopt a stable structure which is closely related to their function. However, since 1980’s more and more proteins have been found to lack such a rigid structure and to reveal only transient secondary structure. Today it is estimated with bioinformatics methods, that in eukaryotic organisms such intrinsically disordered proteins (IDPs) constitute approximately 25-30% of all proteins. Importantly, almost 80% of cancer-related proteins contain a disordered region of at least 30 residues [4]. Moreover, many IDPs are associated with neurodegenerative diseases, as they can form amyloid fibrils. The IDP’s commonness, biological significance and connection with human diseases make them important objects for research.

X-ray crystallography, being an undeniable leader in structural research of folded proteins (over 88% of all protein structures deposited in Protein Data Bank were determined with this method), cannot be used in the case of IDPs, as such proteins are impossible to be crystallized. In contrast, nuclear magnetic resonance (NMR) is a technique enabling studies of both structured and unstructured proteins. First step of an NMR investigation is so-called resonance assignment: to each spectral peak a corresponding spin system (a nucleus or a group of nuclei) should be assigned. Coordinates of the peak correspond to the frequencies of spin precession of nuclei of the spin system. Assignment of resonances is a crucial step in NMR analysis; any errors at this stage will influence further results. In the case of small (up to 20 kDa) folded proteins three-dimensional (3D) spectra are usually sufficient for obtaining complete or almost complete assignment. Nonetheless, decreasing level of ordered structure makes such techniques insufficient. High conformational dynamics of IDPs causes averaging of local magnetic field and consequently – low chemical shift dispersion. In 3D spectra of IDPs the peaks usually strongly overlap. Therefore, higher dimensional techniques should be employed.

Many efforts were put into development of protein-aimed NMR techniques of high dimensionality. Usually, proton excitation and detection is utilized, due to the highest sensitivity of such approach (connected with the highest magnetogyric ratio of proton with respect to other nuclei typically used in protein NMR: ¹³C and ¹⁵N). However, in the case of proline-rich proteins, such techniques often fail. Proline is the only amino-acid residue which does not have an amide proton (HN). Thus, there are no signals originating from proline residues in HN-exited nor HN-detected experiments. Presence of such a residue brakes the chain of sequential connectivities during resonance assignment process. In the case of folded proteins, where proline residues constitute statistically 5.1% of all residues, such gaps usually do not prevent from getting almost complete resonance assignment. However, this is not the case with IDPs. Here prolines – as disorder-promoting residues – are much more common. Stretches of two or three prolines are frequently found. Known are IDPs containing over 25% of this residue in a sequence. Consequently, the length of peptide chain between two proline residues (in which the sequential connectivities can be established using HN-excited and -detected techniques) often does not exceed just a few residues, which makes it difficult to map this chain onto the protein sequence. In such a situation the experimental techniques utilizing excitation and detection of aliphatic protons and/or heteronuclei are favorable.

In recent years, a significant progress in NMR instrumentation was observed. The magnetic fields achieved in spectrometers were increased (currently even 1 GHz spectrometers are available) and cryogenically cooled probes became much more popular. These developments allowed to improve sensitivity of NMR experiments, making possible exclusively heteronuclear experiments. Several ¹³C-detected NMR experiments for proteins have already been proposed. Although some of these experiments were reported to be suitable for studying of IDPs, these were initial studies within this area and there is still a lot of space for advancement.

The project aims at the development of a number of high-dimensional ¹³C-detected NMR techniques dedicated for resonance assignment of IDPs, including proline-rich IDPs. These efforts should result in an unprecedented compression of the acquisition times required by NMR studies of IDPs. The successful implementation of this project will provide the tools for NMR-based structural studies, attractive for the scientific community.

Cooperation

The project is realized in cooperation with Prof. Isabella Felli (Magnetic Centre CERM, University of Florence, Italy) and Prof. Robert Konrat (Institute of Biomolecular Structural Chemistry, University of Vienna, Austria).