PNNL-13501 - Pacific Northwest National Laboratory

Study Control Number: PN00077/1484
Rapid NMR Determination of Large Protein Structures
Paul D. Ellis, Michael A. Kennedy, Robert Wind
Functional and structural genomics is the science that correlates protein structure with function in living cells, and
proteomics is the study of the entire complement of proteins expressed by a cell. This project seeks to develop new
methods of nuclear magnetic resonance (NMR) analysis of large protein structures so that data collection times for
analyses are significantly reduced, allowing more proteins to be studied.
Project Description
Dielectric losses associated with biological NMR samples
at high magnetic fields (>17 T) ultimately determine the
potential NMR signal to noise (S/N) ratio. These losses
have a direct effect of lengthening (quadratically) the time
needed to acquire NMR data sets required to deduce the
three-dimensional structure of a protein in solution. The
goal of this research is to develop technical means for
minimizing dielectric losses in biological NMR
experiments. If successful, this could lead to a significant
reduction in NMR data collection times leading to more
rapid structure determinations. One of the most
innovative new ideas for reducing the dielectric losses in
biological NMR samples is to encapsulate proteins in
reverse micelles dissolved in low dielectric bulk solvents.
Preparing and characterizing such systems by small angle
x-ray scattering and NMR spectroscopy will be the
primary focus of this project.
Introduction
In the post-genomic era, the scientific focus is changing
from determining the complete DNA sequence of the
human genome to characterizing the gene products. The
expected number of proteins from the human genome is
~100,000. Proteomics, defined as the study of the entire
complement of proteins expressed by a particular cell,
organism, or tissue type at a given time for a given
disease state or a specific set of environmental conditions,
promises to bridge the gap between genome sequence and
cellular behavior. An integral challenge involves the
characterization of the biological functions of these
proteins and analysis of the corresponding threedimensional
structure. This is referred to as functional
and structural genomics, respectively. Ultimately, the
goal of structural genomics is to determine the structure
of a sufficiently large subset of the approximately
100,000 proteins such that a “basis” structure could be
formulated. This basis would, in turn, be used as a
predictor of the remaining structures while simultaneously
providing a rationale for the observed function of all of
the gene products.
Currently, x-ray crystallography requires diffractionquality
crystals, selenium-labeled proteins, and access to
high-intensity light sources available at the DOE national
laboratory synchrotrons. Given these conditions, x-ray
data sets can be collected in a matter of 2 to 4 hours and
virtually have no limitation with respect to protein size.
However, dynamic or statically disordered regions of
proteins are invisible to x-ray crystallography. NMR
spectroscopy has several unique capabilities that are
complementary to x-ray crystallography: 1) proteins can
be examined under physiological solution-state
conditions; 2) dynamic regions of the proteins can be well
characterized; 3) intermolecular complexes can be easily
studied as a function of pH, ionic strength, etc. However,
NMR currently has two significant limitations when
compared to x-ray crystallography: 1) data sets currently
take about 60 days to collect, and 2) the size of proteins
amenable to NMR structure determination is currently
limited to ~50 kDa.
If the structures of a large subset among approximately
100,000 proteins must be determined, then x-ray
crystallography data collection would require maximally
between 24 and 45 years of synchrotron time (assuming
proteins protein crystals are obtainable at high enough
quality in all cases). On the other hand, for this same
subset, given the current technology, NMR spectroscopy
would require approximately 16,438 years of NMR time.
Estimates of the cost per structure with current
technologies are running around $50,000 to $100,000
each. The cost of such an effort would be between
$5 billion and $10 billion. Ideally, one could imagine
dividing the task of structural genomics equally between
x-ray crystallography and NMR, but, at the current state
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