Structural biology

The solution structure of a protein/domain of a protein is easily solved given that there is a well-defined three-dimensional structure. Proteins which are only partly folded can be studied combining classic structure determination and relaxation measurements to gain insight in function.

Sample requirements would be >0.4 mM protein concentration in at least 120 µl, and that the protein is stable over a time period of at least three days, preferably more than two weeks.

CSP is a straightforward method to study the interaction of a protein with something present in solution. All kinds of interactions can be monitored – from interactions with small molecules to binding to a large molecular complex such as a photosystem.

Sample requirements are 25-100uM in at least 120 µl using the buffer conditions mentioned above in the general section.
 

The core of protein function often lies in its dynamic properties. The interactions in the cell, performing catalysis or transporting ligands/ions are all feasible because the ability of the protein to adapt its conformation to the particular function.

Sample recommendations is to use concentrations above 0.5mM to ensure a high signal to noise.. 
 

Both relaxation measurements and CSP measurements requires that the backbone of the protein is assigned. This in turn requires ¹³C and ¹⁵N (and for larger proteins, ²H) labelled material. The method for assigning the backbone atoms have been optimised in-house to also include monitoring of the progress during data recording as well as on-the-fly analysis.

Important for successful backbone experiments is to have a sample with relaxation properties ensuring that information about also Cbeta is detected. If this is not the case in the standard situation (¹³C/¹⁵N labelling, 25°C), this can be overcome by either deuterium labelling and/or increase of temperature. 
 

FBS is a methodology to observe a mixture of small molecules, with or without the presence of  a protein or other macromolecule, to find molecules which interact with the protein. For any potential interaction hits, measurements can also be done in the reverse mode, observing the protein instead, not only verifying positive binding but also providing detailed information about the interaction surface by studying CSP, see above.

For screening purposes, the protein is at a low concentration during measurements (10-20uM). The protein needs to be able to tolerate small amounts of dmso since compounds are often dissolved in DMSO at high concentration, and 1-2% DMSO is therefore present in the final NMR sample. For CSP measurements, see above.
 

For proteins with special requirements for labelling or inherent properties (toxicity for example) that prohibits expression in vivo, we offer an in-house cell-free protein expression system through the Protein Production Sweden distributed infrastructure.