The general research interests of our group are to study fundamental mechanism that governs biological process from structural biology point of view. We study the systems that include nucleic acids, enzymes and RNA-protein-drug complexes using NMR and various other biophysical and biochemical methods. Our current research focuses are the following.1. RAP project. Of the more than 140,000 genes in the human genome, roughly 20-30% code for integral membrane proteins. These proteins have a vast array of functions ranging from receptors of cellular signals to channels for transporting ions and small molecules. Most importantly, membrane proteins represent the cellular targets for approximately 60% of all drugs currently manufactured. The low density lipoprotein receptor-related protein (LRP) is a large endocytic receptor that involved in several biological pathways, including Wint pathway, and plays prominent roles in lipoprotein metabolism and in the catabolism of proteinases involved in coagulation. LRP is also the cellular entry gateway for several viruses and toxins. The protein that is responsible for the well-being of this important class of the receptor is called the receptor associated protein (RAP). In the ER, RAP acts like a molecular chaperone by interacting with newly synthesized receptor to help it fold. In the meantime, RAP serves as an antagonist to prevent other ligands from binding to the receptors prematurely in the secretory pathway, and escorts the receptors from the ER to the Golgi where it dissociates from the receptors before recycling back to the ER.
The aim of the project is to study the structure biology of RAP and its interaction with LRP. The first phase of the project is to determine the solution structure of the protein and is to reveal the structure basis for RAP's biological function using various methods and tools. At this point, we are completing the first phase study. We are also planning study the complex structure formed between RAP and the LRP fragment.2. L11 and L11-rRNA-thiostrepton project. High resolution X-ray crystal structures of ribosome together with the accumulative knowledge from previous investigation have tremendously enhanced our understanding the structure and the function of this protein synthesis machinery. In addition to its central role in protein synthesis, ribosome biogenesis and translation control are also essential cellular processe. Several tumor suppressors and proto-oncogenes have been found either to affect the formation of the mature ribosome or to regulate the activity of proteins known as translation factors. Furthermore, several ribosome proteins have been implicated in inactivating HDM2/MDM2, a p53 inhibitor and inducing p53 activity in response to cellular stress. The findings revealed a new pathway of regulating HDM2/p53 activity under stress cellular environment. These newly revealed roles for ribosome proteins necessitate a need to study these proteins from a fresh angle in the context of both ribosome protein synthesis as well as their potential regulatory roles. L11 is recognized to play an important role in the elongation and translocation cycle of protein synthesis, during which the activities of EF-TU and EF-G at both GTP and GDP stages require coordinated movement between the factors and the L11 N-terminal domain. L11 is absent from the 2.4 resolution X-ray map of 50 S subunit, and its orientation between the two domains in the cryo EM density map of 50 S ribosome is different by ca. 40 degrees from that in the binary complex formed between L11 and rRNA. Hence, there is a need for a clear understanding of the L11 structure and its role.
The aim of the project is to study the dynamics of L11 in free and the ternary complex forms, and the mechanism of inhibition of protein synthesis by thiostrepton.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010379-06
Application #
7291757
Study Section
(SBL)
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2005
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Lee, Donghan; Walsh, Joseph D; Mikhailenko, Irina et al. (2006) RAP uses a histidine switch to regulate its interaction with LRP in the ER and Golgi. Mol Cell 22:423-30
Walsh, Joseph D; Wang, Yun-Xing (2005) Periodicity, planarity, residual dipolar coupling, and structures. J Magn Reson 174:152-62
Walsh, Joseph D; Kuszweski, John; Wang, Yun-Xing (2005) Determining a helical protein structure using peptide pixels. J Magn Reson 177:155-9
Wu, YiBing; Migliorini, Molly; Walsh, Joseph et al. (2004) NMR structural studies of domain 1 of receptor-associated protein. J Biomol NMR 29:271-9
Walsh, Joseph D; Cabello-Villegas, Javier; Wang, Yun-Xing (2004) Periodicity in residual dipolar couplings and nucleic acid structures. J Am Chem Soc 126:1938-9
Cabello-Villegas, Javier; Giles, Keith E; Soto, Ana Maria et al. (2004) Solution structure of the pseudo-5' splice site of a retroviral splicing suppressor. RNA 10:1388-98
Neamati, Nouri; Murthy, Manisha; Wang, Yun-Xing (2003) Preparation of DNA-protein complexes suitable for spectroscopic analysis. Methods Mol Med 85:185-202
Wu, YiBing; Migliorini, Molly; Yu, Ping et al. (2003) 1H, 13C and 15N resonance assignments of domain 1 of receptor associated protein. J Biomol NMR 26:187-8