核磁共振在蛋白质研究中的作用

Proteins are key to a vast range of diverse functions within the body, from muscle contraction to immune response. Be it a structural or dynamic role that the protein plays, it will have a highly specific structure that enables it to perform its assigned task perfectly. Much of our understanding of biological systems has thus arisen from research into the structure of, and interaction between proteins.

Nuclear magnetic resonance spectroscopy (NMR) is a sophisticated research technique that provides detailed information about the structure, dynamics, reaction state, and chemical environment of molecules without damaging the sample. It is ideally suited to investigating the structure of proteins, conformational changes in proteins and chemical kinetic processes.

In a recent interview, Fábio Almeida, discussed the role of NMR in his research investigating protein structure and dynamics. Fábio works in the National NMR Center at the Federal University of Rio de Janeiro, a division of the Center of Structural Biology and Bioimaging.

Role of NMR in Protein Research

There are three key methodologies used in the study of protein structure —x-ray crystallography, cryomicroscopy, and NMR — but NMR is the only one that enables proteins to be studied both in solution and in solid state. It also makes it possible to measure the dynamic properties of each individual nuclei in the protein, which is very important as these dynamics are intimately related to the biological mechanism of the protein.

Fábio summarised “NMR is very special in the sense that you can study the structure, but also the dynamics of protein, and in this sense NMR is unique”.

Multi-domain Proteins

Recently, structural studies of higher molecular complexes have been enabled by the development of cryo-electron microscopy. Complemented with the insight that NMR provides on protein dynamics, the duo of technologies has contributed significantly to understanding the motion of multi-domain proteins. In addition, NMR studies have helped elucidate the intrinsically disordered segments of protein (which are encoded by 30% of the entire genome) that provide linkers between the different domains in multi-domain proteins, and also sometimes in smaller proteins too. Since these linkers contribute to the entropy of a system, they also are very important in biological mechanisms.

Thioredoxin

Thioredoxins are small redox proteins present in all organisms that are involved in many important biological processes. Although the structure of thioredoxin was elucidated many years ago, there are still dynamic and structural elements that need to be clarified. Recently, Fábio and his team were able to describe one of these dynamic elements; they showed that the water cavity is essential and very important for the biological mechanism of thioredoxin.

He has also used NMR to study transient oscillation in thioredoxins of higher eukaryotes. During their more recent evolution, thioredoxins have gained the ability to undergo post-translational modifications, such as transient oscillations and dilations. NMR has allowed the mechanisms by which this occurs to be studied.

Oncogenes and Viruses

Protein studies are important for helping inform strategies to control human pathogens since in the majority of cases pathogenesis is achieved through the interaction of proteins and nucleic acids. NMR facilitates such study by enabling properties at the atomic level to be observed directly and measured.

Fábio Almeida commented “For proteins, all these biological macromolecules, proteins, carbohydrates, and nucleic acids, NMR is one of the main methods to approach and to ask biological questions… NMR is very good to measure interactions and to measure the role of dynamics in these interactions. I believe NMR is very powerful in this sense”.

For example, NMR has been used to describe the binding mechanism of dengue virus capsid proteins to intracellular lipid droplets that play a part in lipid metabolism. This binding is essential to the virus’ survival, so by mapping this interaction through NMR analysis, it is hoped that new compounds can be developed to prevent the binding of the dengue virus capsid protein to lipid droplets and provide a treatment for Dengue fever.

Similarly, NMR has enabled determination of the structure of Zika virus capsid proteins, and is currently being used to study the interaction of Zika virus capsid proteins with intracellular molecules with the aim of uncovering its biological mechanism of action.

NMR spectroscopy has also advanced knowledge of cancer biology, and guided the development of new oncology treatments. For example, NMR was pivotal in understanding the biological role and the structure and dynamics of the oncogene p53. Oncogenes are proteins involved in the formation of tumours, usually by causing dysregulation of the cell cycle. Mutations in the protein p53, a tumour suppressor, give rise to 50% of all cancers.

Preparing a Microbiological Sample for NMR Analysis

In order to study a protein of interest, the gene encoding it must first be cloned. This is usually achieved in a bacteria. The protein can then be expressed and labelled with an isotope, such as nitrogen 15, carbon 13, or deuterium, that has magnetically active nuclei to enable NMR measurements.

The purified, labelled protein can then be studied under physiological conditions to assess the effect of different osmolytes, and changes in pressure. The same sample can be subjected to different conditions to see how they impact the structure and dynamics of the protein. Such biological assays allow the ideal working condition of the target protein to be determined.

Criteria for Choosing New Instrumentation

Fábio Almeida also highlighted the importance of choosing the correct instrumentation to suit the kind of studies for which it is intended.

The first choice to be made is the required NMR field. Is the extra cost of a high field instrument justified or will a lower field spectrometer be adequate? It may be that multiple NMR fields will be required if dynamic studies are to be performed.

Secondly, it is necessary to select the most appropriate type of console that provides an adequate number of channels for the proposed studies. The more nuclei that need to be measured, the more channels are needed. It is also important to choose the correct level of power; if very short pulse are likely to be needed, higher power equipment will be required.

Fábio explained “Here, for instance, all of our spectrometers have four channels, because we measure proton, nitrogen 15, carbon 13, and deuterium…but not everyone will need four channels”.

It must also be ensured that the instrument has the appropriate level of specifications, such as magnetic field stability, pulse field stability.

A final important consideration is the ease with which the instrument can be maintained, especially if the country where the instrument is produced is not close by. For example, Brazil is far from the biggest centres in Europe and the United States so it is important to check that the company from which the instrumentation is sourced has a support team in Brazil who are able to solve any problems. Having a good partnership with the company from which the spectrometer is bought, with reliable customer service and technical support is a key consideration when buying new instrumentation.

Having taken all these factors into account, the NMR center at the Federal University of Rio de Janeiro selected Bruker NMR instrumentation.

Fábio explained “We have six NMR spectrometers from Bruker with several fields. Bruker has several, it provides good quality equipment. Magnets with high homogeneity and stability, low use of helium…and we have access to the Bruker engineers”.