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Research Projects

The primary aim of the Laboratory for Biomolecular NMR Spectroscopy is to study structure and dynamics for biological macromolecules using a combination of solid- and liquid-state NMR spectroscopy. This research represents an integral part of a large activity at the University of Aarhus using a combination of protein chemistry, gene expression, macromolecular X-ray diffraction, and NMR spectroscopy to study structure/function relationships for proteins. The NMR work covers in particular peptides/proteins immobililized by polymerization, aggregation, or membrane association. To achieve this goal the research covers methodology development, experiment setup/implementation on model samples, and applications involving studies of, e.g., membrane proteins (ion channels), active metal binding sites, biomineralization, colloid formation etc.

The following sections exemplify these aspects by describing some recent research projects of the group. Although described separately, the methodology and application project represent an unity upon which the coming research of the laboratory is based.

NMR Methodology

An important research activity is the development of multiple-pulse solid- and liquid-state NMR experiments providing optimum conditions for obtaining information about structure and dynamics of biological macromolecules. This research covers all aspects of experiment design ranging from development of theoretical quantum mechanical tools through construction of advanced computer software and experimental hardware to experimental implementation on model samples and real systems. Based on a systematic strategy for experiment design the methodology work focus on construction of "complete experiments" as well as important "building blocks", providing, e.g., selective evolution under certain parts of the Hamiltonian or optimum physical conditions for the experiment.

Development of Multiple-pulse Solid-State NMR Experiments

The solid-state NMR methodology work primarily focus on the design of efficient methods which through appropriate high-order re- and decoupling of anisotropic nuclear spin interactions tailor the Hamiltonian to accomplish optimum coherence transfer in complicated nuclear spin systems. Recently, this has lead to the design of pulse sequences for homonuclear dipolar recoupling and 2Q filtration (e.g., rotary resonance, HORROR, C7, POST-C7), heteronuclear dipolar recoupling (e.g., GATE), combined dipolar and anisotropic shielding recoupling (e.g., MSD-HORROR, rotary resonance type experiments), homonuclear dipolar decoupling (e.g., MSHOT-3, HOT-FSLG), and spectral editing (e.g., CPD and CPDR). A variety of these experiments represents important building blocks in pulse sequences for solid-state NMR structure determination of peptides/proteins in rotating and uniaxially oriented samples. The most recent methods have been designed using new concepts for high-order average Hamiltonian theory (the semi-continuous Baker-Campbell-Hausdorff (scBCH) expansion) and an advanced computer simulation package (SIMulation Package for SOlid-state Nmr spectroscopy: SIMPSON) developed in this laboratory. For integer quadrupolar nuclei we have recently demonstrated new variants of 2D MAS satellite transition NMR experiments (2H) developed using an advanced Lie algebra formalism. For half-integer quadrupolar nuclei with large quadrupole couplings we have recently introduced a series of new pulse sequences exploiting QCPMG sampling of the signal to improve the sensitivity of quadrupolar-echo experiments by more than an order of magnitude. These experiments (QCPMG, QCPMG-MAS, MQ-QCPMG-MAS) open up new possibilities to study the structure and dynamics for, e.g., metalloproteins.

Development of Liquid-State NMR Pulse Sequences

A considerable part of our research activities focus on systematic development of optimum pulse sequences for liquid-state NMR studies of proteins. The methodology development is based on unitary bounds on spin dynamics (which provides information about the maximum transfer efficiency for given coherence transfer processes and the unitary propagator, i.e., the experiment accomplishing this) as well as numerical and analytical approaches for systematic experiment design. The latter involves software which using a gradient based approach determines the unitary bound for the relevant coherence transfer process and software which based on this information in an iterative fashion designs the optimum experiment. Using this setup (analytical or numerical) it is straightforward to test whether existing (if any) experimental procedures are optimal and if not design experiments providing the largest possible coherence transfer with the desired selectivity concerning initial and target spin operators. That this method is generally applicable has recently been demonstrated, for example, by the development of INADEQUATE CR (which double the sensitivity relative to the popular INADEQUATE experiment) and in- and antiphase COS HSQC experiments for sensitivity optimized Coherence Order Selective (COS) heteronuclear coherence transfer in IS, I2S, and I3S spin systems. As another example, we have recently developed Coherence-Order- and Spin-State-Selective (COS3) HSQC experiments providing the theoretical maximum transfer efficiency as well as improved spectral resolution by transferring all coherence into one of four spectral lines.

Software Development

The construction of efficient numerical procedures for efficient simulation of solid- and liquid-state NMR pulse experiments and spectra represents a fundamental part of our research. This applies both to the methodology projects described above but also to the more application-oriented projects as a tool for extraction of structural parameters from solid-state NMR spectra and for the relation of these to molecular structure and dynamics. Besides a large number of specialized simulation programs and procedures for fast calculation of solid-state NMR powder spectra, this has recently led to the software package SIMPSON (SIMulation Package for SOlid-state Nmr spectroscopy) which represents a flexible "computer spectrometer" enabling simulation of essentially all types of multiple-spin, multiple-pulse, multiple-dimension solid-state NMR experiments and their associated spectra.

Development of Quantum Mechanical Tools

Another important element in our strategy for systematic design of optimum NMR methods is the development of quantum mechanical tools which provide analytical information about details of the spin dynamics. This information can be the effective (i.e., time independent "average") Hamiltonian for a given pulse sequence (or pulse sequence element) or information about the theoretical maximum efficiency for a given coherence transfer process (or for the scaling factor for the effective Hamiltonian). In this context we have have recently introduced the "the semi-continuous Baker-Campbell-Hausdorff (scBCH) expansion" to calculate the effective Hamiltonian for multiple-pulse experiments to high order and had a number of projects concerning "unitary bounds on quantum dynamics" which determine the theoretical upper limit for any relevant coherence or polarization transfer process.

Hardware Development

The group has been involved in several hardware development projects enabling specific solid-state NMR experiments to be performed under optimum conditions. This involves, for example, design of solid-state NMR probes with resonator coils to improve rf inhomogeneity and flat-coil NMR probes with effective hydration control for studies of peptides/proteins uniaxially oriented in phospholipid bilayer membranes.

Application Projects

Membrane Peptides/Proteins.

Using the homebuilt flat-coil NMR equipment, several of the pulse sequences described above, and the software for analyzing spectra in terms of restriction plots (SIMPSON) we have applied various one- and two-dimensional solid-state NMR experiments to study membrane association of 15N isotope labeled peptides acting as ion-channels or as membrane anchors for larger proteins. As a typical example the latter includes, the C-terminal part of PP3 for which we have determined the structure in organic solution, SDS micelles, and the conformation in phospholipid bilayer membranes using a combination of liquid-state NMR, CD spectroscopy, and solid-state NMR spectroscopy. Current research projects concern various ion-channels peptides/proteins being studied by appropriate combinations of solid- and liquid-state NMR spectroscopy.

Determination of anisotropic interaction tensors for peptides

Recently there has been a considerable interest in exploiting anisotropic chemical shielding and dipolar coupling tensors in structure determination of proteins. This applies obviously to solid-state NMR, but also to liquid-state NMR where it has become very popular to establish residual effects from these anisotropic interaction by dissolving the proteins in bicelle or phage solutions. We have recently introduced and applied several new methods using re- and decoupling methodology to obtain information about the magnitude and orientation of 1H, 13C, and 15N chemical shielding and associated dipolar coupling tensors from MAS experiments of amino acids and peptides in powder samples.

Metal binding sites

Using our recent QCPMG and QCPMG-MAS experiments for sensitivity-enhanced quadrupolar-echo spectroscopy we have in several projects focused on the coordination of metal cations to ligands in metalloproteins and model-compounds. For examples, we have recently studied the 67Zn metal coordination (in terms of electric field gradients) in organometal compounds serving as models for proteins such as thermolysin and carboxypeptidase A.

Colloids and biomineralization

In various of projects we have recently used 31P MAS solid-state NMR spectroscopy to examine structural aspects of biomineralization and biological colloid formation. For example, we have studied casein micelles (bovine, caprine, ovine), caseins, bone material, renal calculi (urinary and bladder stones), and a large variety of inorganic amorphous/crystalline calcium phosphates. In these studies we have focused on the role of phosphorylated serine residues in the proteins and inorganic calcium phosphates with respect to structure and composition.

Various different application projects

During the past few years we have performed a number of application projects relating to other areas of science. This involves, for example, determination of structure and dynamics for organic polymers, inorganic compounds, and oil-source rock and clay minerals.