The molecular mechanisms that mediate the association of ubiquitin chains are complex and not fully understood. For example, it is not clear how the atypical K27-, K29- and K33-linked diUbs can form a close association despite their distinct sequence and spatial positions. The functional landscape of these diUbs can be investigated by studying the conformational distribution and free energy surfaces of MD trajectories.
Synthesis
In order to comply with the principles of green chemistry, we have designed an efficient synthetic route based on the condensation of acrolein and tert-butyl acetate to generate the unique precursor acetoxyester. This is converted into a series of (di-)lipidic HAAs by cross-metathesis with hept-1-ene (Scheme 3).
In spite of seven chemical steps in total, PMI ratios are low and the overall process is more sustainable than previous routes to (di-)lipidic HAAs (Bauer et al., 2015). Furthermore, the route is scalable up to the kilogram scale, using common equipment and materials, without any need for anhydrous solvents.
Acrylonitriles are a key building block in many industrial processes such as the production of acrylic fibers and polyacrylonitriles. Moreover, they are also found in natural products and pharmaceuticals including Entacapone (anti-Parkinson's disease drug), CC-5079 (an anti-tumor drug) and Rilpivirine (anti-retroviral drug). Efficient synthesis of acrylonitriles is therefore a long-standing challenge in organic chemistry. Our new protocol works under mild conditions, using a base-promoted tandem nucleophilic substitution-isomerization of cyanohydrin methanesulfonates with alkenyl boronites. This offers high chemical yields and good functional group tolerance.
Purification
Purification is the process of making a substance free of unwanted particles or contaminants. The purpose of the process is to make a material ready for a desired application, such as for use in pharmaceuticals or biotechnology. There are several methods for purifying a substance, including dialysis, desalting, and affinity chromatography. The process of purification can also be used to remove contaminants from the water supply. It can also be used to prepare a sample for a specific analysis.
The process of purification is not always easy. For example, drinking water must be tested for many contaminants, including bacteria, viruses, and fungi. Even natural spring water that was considered safe for human consumption in the 19th century now requires extensive testing and treatment before it can be consumed. It is also difficult to determine what sort of treatment is necessary, and chemical and microbiological analyses are costly.
Our kinetic data suggest that the conformational equilibrium of K6-, K11-, and K48-diUbs is highly pH-dependent, and lowering the pH increases the population of open conformations. The kinetic model for K33-diUb is more complex, however. It has two functional states that are similar to the compact structures of K11- and K48-diUbs. We are investigating the structural mechanisms that distinguish these states. In particular, we are examining the roles of hydrophobic interactions and electrostatic forces in this reaction.
Structural Characterization
The structural characterization of cgk33 was accomplished by NMR spectroscopy and computational modeling. The NMR spectra were recorded at 298 K using a 600 MHz instrument. The carbon signals from residues A to F were assigned by HSQC, combined with literature data and methylation analysis.
The functional landscape of K6-, K11-, K48- and K33-diUbs has been analyzed by free energy surfaces projected on two order parameters independent of their native structures: the distance between I44 hydrophobic patches (1) and the distance between I36 and I73 patch involving L8, I36, and L73 (2). The results indicate that these regions play an important role in the formation of Ub interfaces. Moreover, the simulation results show that the K11-diUb conformational distribution is shifted to a more closed state compared to the K48-diUb conformational distribution and that the symmetry between Ub monomers is preserved in the compact structures of both the K6- and the K11-linked diUbs.