In the cellular environment, biomacromolecules together occupy about 20-30% of the total volume and sometimes up to 40% by estimation. The occupied volume is not accessible for other molecular activities, creating a crowded condition. However, common biochemistry studies are carried out in test-tubes where the buffer solution is diluted. Although these in vitro studies give us a lot of useful information on properties of biomolecules and help us understand their cellular functions, the difference between the molecular behavior in dilute solutions and in crowding solutions are not well defined. In some cases, the difference may be so significant that researchers might be misled. In order to better understand the behaviors of biomolecules in cells, more and more researchers have de...[ Read more ]

In the cellular environment, biomacromolecules together occupy about 20-30% of the total volume and sometimes up to 40% by estimation. The occupied volume is not accessible for other molecular activities, creating a crowded condition. However, common biochemistry studies are carried out in test-tubes where the buffer solution is diluted. Although these in vitro studies give us a lot of useful information on properties of biomolecules and help us understand their cellular functions, the difference between the molecular behavior in dilute solutions and in crowding solutions are not well defined. In some cases, the difference may be so significant that researchers might be misled. In order to better understand the behaviors of biomolecules in cells, more and more researchers have developed an interest in the effects of the crowded intracellular environment. In this thesis, we studied crowding effect on selected proteins’ function, stability and fast dynamics.

Firstly, we studied the crowding effect on CalE8, the polyketide synthase (PKS) in the biosynthesis pathway of natural products calicheamicin. Previous studies in our research group have already found that a crowding agent can suppress the formation of side-products formed by a two-module nonribosomal peptide synthetase (NRPS) in the biosynthesis pathway of enterobactin. Polyketide synthases are a group of enzymes very similar with nonribosomal peptide synthetases. Here we chose this small PKS CalE8 as a model enzyme and the results showed that crowding agents would not improve the activity and specificity of CalE8. This further demonstrates the complexity of crowding effect on enzyme activity.

Subsequently, we studied the crowding on protein stability where calmodulin was used as a model. Previously researchers have already found that the crowding agent Ficoll 70 can improve the thermal stability of calmodulin. In our study, the stabilization effect of Ficoll 70 on calmodulin thermal stability was also observed. Further experiment showed that Ficoll 70 could also stabilize protein chemical stability. Meanwhile, sucrose, the monomer of Ficoll 70 which is structurally similar with Ficoll 70, could improve calmodulin thermal and chemical stability to a similar extent. Considering that sucrose is a small molecule without crowding effect, these results strongly indicate that some previously reported crowding effect is mainly from the solvent effect introduced by high concentration of crowding agent.

Finally, we studied the crowding effect on dynamics of calmodulin, which is saturated with calcium ions. Steady state fluorescence was used to monitor the signal of two tyrosine residues at C-terminal domain of calmodulin. At elevated concentration of Ficoll 70, the emission at about 307 nm continuously decreased. In 30% Ficoll 70 solution, the tyrosine signal decreased to as much as about 40%. In comparison, sucrose could not affect calmodulin fluorescence. This Ficoll 70 induced signal decrease was very similar to the fluorescence change at elevated temperature which indicated fast dynamics increase of the two tyrosine residues. The calmodulin backbone dynamics was measured by NMR in normal buffer as well as 18% sucrose and Ficoll 70 solutions. The order parameter (S²) obtained from model-free analysis showed that most of the protein residues had a larger S² value in Ficoll 70 solution than in sucrose solution or normal buffer. That is to say, the whole protein backbone was getting very rigid after being transferred into crowded environment. In sum, we showed the first experiment evidence of crowding effect on protein backbone fast dynamics decrease and side-chain fast dynamics increase.