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Shan Group

Shu-ou Shan

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Sowmya Chandrasekar

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Project description: Sowmya is exploring a recently discovered novel SRP system from chloroplast (cpSRP). The cpSRP is one of the machineries that targeting light harvesting chlorophyll proteins and complexes from the lumen of chloroplast to the thylakoid membrane. Intriguingly, this system uses a novel protein, cpSRP43, in place of the otherwise universally conserved SRP RNA, and employs a post-translational mode of targeting in place of the co-translational mode. Thus, the cpSRP system offers a unique opportunity to probe the role of SRP RNA (or cpSRP43) in protein targeting. We will define the energetic features of the GTPase cycles in the cpSRP system. We will compare the role of cpSRP43 in the GTPase cycles of cpFfh and cpFtsY to that of the SRP RNA in classical systems, and search for conditions that allow the SRP RNA and cpSRP43 to cross-complement. These experiments will reveal the integral functional role of the molecules that are conserved among the different pathways.

The different mode of substrate utilization in the cpSRP pathway can help us understand the coupling between the targeting reaction and the GTPase cycles of SRP and SR. We will compare the mode of SRP-substrate interaction in the chloroplast system, which lacks a translating ribosome, with the canonical SRP systems that recognize ribosomeonascent chain complexes. Further, because of the simplicity of the cargo protein the cpSRP system will provide a simpler, more accessible system to develop quantitative in vitro targeting assays. Thus, the cpSRP pathway offers a complementary and conceivably much faster route to the elucidation of the mechanism by which the GTPase cycles of SRP and SR regulate the protein targeting reaction.

Rumana Rashid

CV

Project description: Rumana is developing in vitro assays that quantitatively measure the targeting efficiency of model cargo proteins. We have already developed a semi-quantitative targeting assay based on signal peptidase cleavage of pre-proteins to yield the shorter, mature form of the protein. In the future, we will develop a fluorescence-based targeting assay that allows each step of the targeting reaction to be followed in real time. To this end, we will co-translationally incorporate environmentally sensitive fluorescent probes, such as NBD, into the signal sequence of the nascent polypeptide. The fluorescence of the probe will change upon interaction of the signal sequence with SRP, upon transfer of the signal sequence to the translocon, or upon formation of other intermediates during targeting. The time course of these fluorescence changes will be analyzed to determine the kinetics of the individual steps and the stability of the intermediate species during the targeting reaction.

With these in hand, we can use our knowledge of the GTPases and the extensive array of mutant proteins to selectively perturb the GTPase cycle, and determine at which specific step(s) the targeting reaction is affected by such perturbations. The results will provide valuable clues to the nucleotide requirements of individual steps along the targeting pathway, and to the contribution of GTP binding and hydrolysis to the efficiency or fidelity of protein targeting.

Xin Zhang

CV

Project description: Xin's is developing fluorescence-based approaches, including FRET and environmentally sensitive fluorescent probes, to detect and characterize the individual conformational steps during the SRP-SR interaction.

The general strategy is to incorporate thio-reactive fluorescent probes at specific cysteine residues on the protein. When placed at proper locations, bimolecular SRP-SR association and subsequent conformational changes within the complex will give rise to fluorescence changes. These fluorescence changes can be followed in real time to order the various steps with respect to each other and to determine the kinetics of the individual steps and the stability of the intermediates. The extensive array of GTPase mutants that block the SRP-SR interaction cycle at specific stages will provide invaluable tools for interpreting the observed fluorescence signals and understanding their functional significance.

The results will establish a framework for the GTP-dependent SRP-SR interaction cycle that incorporates not only thermodynamic and kinetic, but also structural information. This will then allow us to determine how other factors contribute to the SRP-SR association and activation. For example, the SRP RNA has an unprecedented catalytic role in the SRP-SR interaction, accelerating both the association and dissociation rate constants by 400-fold. How does the SRP RNA provide such a catalytic role? Analysis of the RNA dependence of the individual steps during the SRP-SR interaction will allow us to determine whether the RNA facilitates the initial bimolecular association or a conformational step at a later stage, and suggest the molecular mechanism by which the RNA achieves the rate acceleration. Similar approaches can be used to determine how other components in the protein targeting reaction, such as the cargo protein and the membrane translocon, affect the SRP-SR interaction cycle