Fast-pulsing longitudinal relaxation optimized techniques: enriching the toolbox of fast biomolecular NMR spectroscopy. Prog Nucl Magn Reson Spectrosc 55: 238-265.
Nuclear magnetic resonance (NMR) spectroscopy is the only biophysical method besides X-ray crystallography that can provide high-resolution structures of biological molecules such as
proteins and nucleic acids and their complexes at atomic resolution. The first NMR-derived three-dimensional solution structure of a small protein was determined in 1985, which means that NMR
is thus about 25 years younger than biomolecular X-ray crystallography. Since then, significant improvements have been made
in NMR hardware (magnetic field strength, cryoprobes) and NMR methodology, combined with the availability of
Availability of molecular biology and biochemical methods for preparation and isotopic labeling of
isotopic labeling of recombinant proteins have facilitated the use of NMR to characterize the
structure and dynamics of biological molecules in solution. These improvements are ongoing and
They aim to overcome the two main problems of NMR of biomacromolecules, namely signal-to-noise ratio and spectral overlap. Importantly, biomolecular NMR spectroscopy can provide information
about conformational dynamics and exchange processes of biomolecules on time scales from
from picoseconds to seconds, and is very efficient in determining ligand binding and mapping the
Interaction surfaces of protein/ligand complexes.
The nuclei of the naturally occurring atomic isotopes that make up biological molecules have either a
nuclear spin in natural abundance (1H, 31P) or naturally less abundant isotopes with a nuclear spin
(13C, 15N) can be incorporated into biomolecules by isotopic labeling. Nuclear spin is
Nuclear spin is associated with a magnetic moment required to achieve nuclear magnetic resonance, and
defines the fundamental resonance frequency, i.e., 600/150 MHz for 1
H/13C at 14.1 Tesla. Each spin in a
molecule leads to a nuclear magnetic resonance line. The exact resonance frequency depends on
The exact resonance frequency depends on the chemical environment of the respective spin, so that, for example, the NMR spectrum of a protein
NMR spectrum of a protein shows NMR signals with slightly different frequencies. These differences are called chemical shifts.
The first step of a structure determination by NMR consists in assigning the chemical shifts of all
of the atoms/spins of the molecule observed in an NMR spectrum. Once the NMR signals are
are assigned, experimental parameters are measured that define the three-dimensional structure.
The most important structural information derived from NMR spectra is based on the nuclear
Overhauser effect (NOE), which results from the crosstalk between different spins (usually
protons) in a molecule and depends on the distance between these spins in space. NOEs are
typically observed only between protons separated by less than 5-6 Å.
Constants mediated by chemical bonds provide information about dihedral angles,
and thus can define the conformations of the peptide backbone and side chains. Recently, new NMR
parameters, such as residual dipolar couplings (RDCs) and cross-correlated relaxation effects.
(CCRs), have been shown to provide distance-independent projection angles for binding vectors, e.g.
N-H and Cα-Hα bonds in proteins. Residual dipolar couplings are found in anisotropic
solution, e.g., in dilute (3%) liquid crysalline media. In particular, RDCs are extremely useful for
determining the relative orientation of two domains of a protein (NOEs between separated domains will be
NOEs between separated domains are often not observed due to the upper distance limit of 5 Å for observing a NOE).
Nowadays, three-dimensional structures can be obtained for proteins up to a molecular weight of 50 kDa.
and NMR spectra can be recorded for molecules well above 100 kDa. In the following
Use and applications of biomolecular NMR in structural biology are presented, basic principles and
fundamentals and observables of biomolecular liquid crystal NMR are described, and the practical aspects are discussed.
To provide an overview of the utility, but also the limitations, of NMR spectroscopy for
structure/function studies of biomolecules.