Jean Baum - US grants

The broad long term objectives are twofold. One is to develop methods that will extend the power of solution NMR beyond the study of native globular proteins to the study of nonnative and nonglobular proteins. The second objective is to use NMR to understand protein folding mechanisms by studying structures of partially folded proteins, and by assessing the effect of amino acid substitutions on structure, stability and folding kinetics. These studies will form the basis for using NMR to understand how interruptions in the Gly-X-Y pattern, found in collagen diseases like Osteogenesis Imperfecta and Ehlers Danlos Syndrome, can result in serious disease. The first aim is to characterize the partially folded state of guinea pig alpha-lactalbumin to elucidate the nature of protein folding intermediates. More specifically, we wish to learn which regions of the molten globule state contain regions of secondary structure and whether tertiary interactions are important in stabilizing these regions of secondary structure. We will characterize the partially folded state by 1H NMR methods, and by isotope labelling and heteronuclear 2D and 3D NMR experiments. Key mutants will be made to assess the effects of sequence change on secondary structure and tertiary interactions of the partially folded state. The second aim is to obtain, for the first time, individual residue assignments and the NMR solution structure of triple helical peptides, to determine the role of individual amino acids in stabilizing the triple helix and to understand how key residues direct protein folding. We will examine the effects, by 1D NMR, of (Gly-X-Y) sequence changes on the amount of triple helix formed, and on the kinetics and thermodynamics of folding. To obtain the solution structure we will first design synthetic triple helical peptides to facilitate the spin system identification process. Then we propose 1H NMR experiments as well as heteronuclear NMR experiments that should allow us to perform sequential resonance assignments, and distinguish inter from intra strand NOE's.

The acquisition of a 600 MHz NMR spectrometer, and the upgrade of the existing Varian VXR-500 to UNITY Plus 500, XL-400 to UNITY Plus 400 and the addition of accessories to the UNITY 500 MHz NMR spectrometers at Rutgers University, and at the University of Medicine and Dentistry (UMDNJ), are crucial for the successful execution of the research projects of the Structural Biology community at these universities. The major users of the NMR facility are: Prof. Jean Baum, Chemistry, Rutgers; Prof. Gaetano Montelione, MBB, Rutgers, and CABM; Prof. Roger Jones, Chemistry, Rutgers; and Prof. Stephen Anderson, MBB, Rutgers, and CABM. In addition, there are many Rutgers and UMDNJ faculty who are using, or planning on using, high field NMR in their research, either independently or via collaborations with the major users. The recruitment of Drs. Montelione and Baum to Rutgers University established multidimensional NMR as a major priority on campus. Numerous investigators at both universities are currently collaborating with these two groups. In addition, Drs. Anderson and Jones have been expanding their own research efforts using high field NMR. As a result, all of the existing NMR instruments are in constant use (24 hrs/day, 7 days/week), and the development of independent NMR programs by other investigators has been prevented because of insufficient instrument time. The lack of a 600 MHz spectrometer on the campus and deficiencies in the existing equipment severely limit research efforts with larger (10 - 30 kD) proteins and complexes. In addition, the NMR instruments used to recruit Baum and Montelione are now out of date. The 500 and 400 MHz spectrometers in the Chemistry Department lack triple-resonance and PFG capabilities which are essential for heteronuclear NMR of macromolecules. None of the instruments are equipped with deuterium decoupling capabilities required to exploit random deuteration techniques for overcoming short 13C relaxation rates encountered in larger proteins. The NMR research and research training of the four major users require direct access to NMR instrumentation for about 40 researchers. In addition, the independent users, mentioned above, have on the order of 40 graduate students and postdoctorals doing 'hands on' NMR experiments. The NMR facilities will also have a significant impact on research and teaching for some additional 40 faculty, students, and postdoctorals at Rutgers and UMDNJ through the research programs of collaborators. In total, approximately 120 scientists will be affected by the proposed NMR hardware upgrades and acquisition of a new 600 MHz NMR spectrometer. The research activities of the major users of the NMR facility are in structural biology with emphasis in the following areas. 1) development of new NMR experiments which facilitate and automate the protein structure determination process; 2) structure determination of small biomedically important proteins (sizes range from 7 to 30 kD); 3) conformational studies of denatured or partially denatured proteins to understand the mechanism of protein folding; 4) atomic details and dynamics of molecular recognition; 5) studies of nucleic acid structure and interactions. In addition, a number of independent users are interested in the analysis of organic and inorganic compounds. The acquisition of a 600 MHz NMR and the upgrades of the existing facility will greatly improve the scientific and engineering infrastructure of Rutgers and UMDNJ. In order to maintain the momentum of this developing dynamic scientific community in structural biology, the NMR facilities must be brought up to the cutting edge of the field.

A grant has been awarded to Rutgers University under the direction of Dr. Jean Baum to purchase an 800 MHz NMR spectrometer as part of an initiative to develop a New Jersey State-Wide 800 MHz NMR Facility. The 800 MHz NMR will initially serve a minimum of four New Jersey institutions including Rutgers-New Brunswick (NB), Rutgers-Newark, Princeton University and Montclair State University (MSU). Ten faculty members, three from Rutgers-NB/RWJ, one from Rutgers-Newark, four from Princeton and two from MSU are participating in the New Jersey Wide 800 MHz NMR Facility. Access to an 800 MHz NMR will allow the State-Wide group to implement a range of state of the art biological NMR experiments that are currently not feasible on the 500 and 600 MHz NMRs and will enhance their productivity in terms of time intensive NMR projects.

Establishment of the NJ State-Wide 800 MHz NMR Facility will enhance the quality and accessibility of NMR research and education to the NJ community. State of the art NMR research which cannot presently be performed on the lower field instruments that exist at the respective individual institutions will now be accessible to the New Jersey NMR community. In addition, the NJ-State wide 800 MHz NMR facility provides an opportunity for undergraduates, graduates and postdocs to meet researchers from other New Jersey schools, to be trained on state of the art NMR instrumentation and to be exposed to many different NMR research projects. Having the 800 MHz NMR serve a number of different institutions provides an intellectual environment and collaborative framework for NMR users. 'Hands on' NMR experiments will be performed by approximately 60 graduate students, postdocs as well as undergraduates involved in NMR research. The research activities of the members of the facility are in molecular biophysics with emphasis in the following areas: Protein structure determination; Conformational studies and dynamics of folded and unfolded proteins to understand the basis of protein folding and misfolding; Studies of nucleic acid structure and interactions; NMR investigations of protein-protein and protein-nucleic acid interactions; Design and conformational analysis of small peptides; NMR methods development for next generation application.

The New Jersey Statewide 800 MHz NMR facility that we will establish will provide infrastructure and enhance the quality and accessibility for many research and educational programs at both private and public Universities in New Jersey. In bringing together this group of Universities, we are establishing an environment of collaboration and are coordinating expertise in NMR from many different institutions. The NJ wide 800 MHz NMR facility provides an opportunity for undergraduates, graduates and postdocs to meet researchers from other New Jersey schools and to be exposed to many different NMR research projects. Having the 800 MHz NMR serve a number of different institutions provides an intellectual environment and collaborative framework for NMR users.

This grant partially supports the acquisition of an 800 MHz NMR cryoprobe to be sited at Rutgers University, Busch science campus. The cryoprobe technology will initially serve a minimum of four New Jersey institutions including Rutgers-New Brunswick (NB), Rutgers-Newark, Princeton University and Montclair State University (MSU). Eleven faculty members, three from Rutgers-NB/RWJ, two from Rutgers-Newark, four from Princeton and two from MSU are participating in the New Jersey Wide 800 MHz NMR Facility. Hands-on NMR experiments will be performed by approximately 60 graduate students and postdocs, as well as undergraduates involved in NMR research. The NMR facility will also have significant impact on research and teaching of additional faculty members in New Jersey through the research collaborations with the faculty and students in the facility.

The research activities of the users of the facility are in molecular biophysics with emphasis in the following areas: Protein structure determination; Conformational studies and dynamics of folded and unfolded proteins to understand the basis of protein folding and misfolding and their associated medical disorders; Studies of nucleic acid structure and interactions; NMR investigations of protein-protein and protein-nucleic acid interactions; Design and informational analysis of small bio-medically important peptides; and NMR methods development for next generation application.