The overall goal of this component is to develop and test new alignment medium for dipolar coupling measurement and new strategies for obtaining structural restraints in large proteins and protein complexes. In the first phase of this component, we will focus on developing DMA nanostructures for weakly orienting protein complexes that are solubilized in harsh buffer conditions. Residual dipolar couplings (RDCs) are crucial experimental restraints in structure determination of large proteins when long-range NOEs are insufficient. RDCs are also very useful in orienting domains within protein complexes. Based on our initial success in making DNA nanotubes that can form stable liquid crystal in the presence of high concentration of detergents, we will establish a robust protocol for aligning detergent-protein complexes. The membrane protein systems to be tested include a number of ~30 kDa a helical, polytopic membrane proteins. We will also test the alignment of the EIF4e-4g complex, an important translation initiation interaction previously studied by Wagner and colleagues. This protein complex can only reach NMR-feasible concentration in the presence of a detergent known as CHAPS. In the second phase, we will develop methods of measuring structural restraints for large helical proteins. Hydrogen bond length has very small variation and serves as extremely stringent distance constraint for defining secondary structures. Measurement of the small 3JNCg couplings is perhaps the most straightforward and unambiguous method for determining protein sidechain xi angles. We will implement the relaxation optimized pulse elements (ROPE) to enable measurement of 3hJNc'across hydrogen bonds and 3JNCg that defines sidechain rotamer for large deuterated proteins. We will also develop 4D NOESY and 13C detection scheme for resolving resonance overlap. For helical proteins larger than 300 amino acids, it is usually not possible to resolve most of the short-range NOEs in the conventional15N-separated NOESY due to strong overlap of amide resonances. However, they can be resolved in a 4D 15N- and 13C'- edited NOESY (NOESY- HNCO). We will work with Jeff Hoch's group to significantly shorten the experimental time of 4D NOESY- HNCO by implementing nonuniform sampling and maximum entropy reconstruction methods so that it can be of practical use. We will also develop 13C-observation strategies for resolving resonance overlap and apply them to the measurements of 15N relaxation rates and RDCs. Harvard Medical School, Boston, MA PHS 398 (Rev. 04/06) Page 236 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): Wagner, Gerhard KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator(s). List all other key personnel in alphabetical order, last namefirst. Name ? eRA Commons User Name Organization Role on Project Chou, James jjchou Harvard Medical School Program Director OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Human Embryonic Stem Cells IEI No D Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://stemcells.nih.gov/reqistrv/index.asp. Use continuation pages as needed. If a specific line cannot be referencedat this time, include a statement that one from the Registry will be used. Cell Line PHS 398 (Rev. 04/06) Page 23# Form Page 2-continued Number the followingpages consecutively throughout the application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Wagner, Gerhard - DETAILED BUDGET FOR INITIAL BUDGET PERIOD DIRECT COSTS ONLY PERSONNEL (Applicant organization only) ROLE ON NAME PROJECT Program James Chou Director Research Kirill Oxenoid Fellow Months Devoted to Project Cal. Acad. Sum. INST.BASE Mnths Mnths Mnths SALARY 1.2 112,480 12 46,668 SUBTOTALS ^ CONSULTANT COSTS EQUIPMENT (Itemize) Component 5 FROM THROUGH 5/1/07 4/30/08 DOLLAR AMOUNT REQUESTED (omit cents) SALARY FRINGE REQUESTED BENEFITS TOTAL 11,248 2,818 14,066 46,668 11,177 57,845 57,916 13,995 71,911 | _ SUPPLIES (Itemize by category) NMR tubes, Shigemi tubes $1,000 Labeled amino acids $ 2,000 Chemicals and disposables $1,000 Precursors for ILV,LV or L labeling $ 4,000 Enzymes for cloning $ 2,000 15NH3,13C glucose &D2O $8,000 PCR primers $ 2,000 20,000 TRAVEL Cost for 2 researchers to attend a relevant conference. 3,000 PATIENT CARE COSTS IMPATIENT OUTPATIENT ALTERATIONS AND RENOVATIONS (Itemize by category) OTHER EXPENSES (Itemize by category) NMR spectrometer use $15,000 15,000 CONSORTIUM/CONTRACTUAL COSTS DIRECT COSTS SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a, Face Page) $ 109,911 CONSORTIUM/CONTRACTUAL COSTS FACILITIES AND ADMINISTRATIVE COSTS TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD $ 109,911 PHS 398 (Rev. 04/06) Page 238 Form Page 4
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