13C, 15N, 2H labeled proteins for NMR structure...

Details 13C, 15N, 2H labeled proteins for NMR structure... 13C, 15N, 2H labeled proteins for NMR structure...

2000-07-10

2002-01-22

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Using a micro-organism to make a protein or polypeptide

C530S300000, C530S350000

Reexamination Certificate

active

06340578

ABSTRACT:

FIELD OF THE INVENTION
This invention is concerned with determining the three-dimensional structure of biological macromolecules, especially proteins. In particular, it is concerned with methods for rapidly determining protein structures by NMR spectroscopy, by providing methods for simplifying NMR spectra using labeled proteins prepared from specifically isotopically labeled amino acids, and the means whereby these labeled proteins and amino acids may be obtained.
BACKGROUND OF THE INVENTION
For many years, there has been intense interest in determining the three-dimensional structures of biological macromolecules, particularly proteins. So called “structure-function” studies have been carried out to determine the structural features of a molecule, or class of molecules, that are important for biological activity. Since the pioneering work of Perutz and coworkers on the structure of hemoglobin (Perez, M. F. et al.,
Nature,
185:416-22 (1960)) and that of Watson and Crick on DNA in the 1950's (Watson, J. D. and Crick, F. H. C.,
Nature,
171:737 (1953), both of which led to the respective scientists receiving the Nobel Prize, this field has been of major importance in the biological sciences.
More recently, the concept of “rational drug design” has evolved. This strategy for the design of drugs involves determining the three-dimensional structure of an “active part” of a particular biological molecule,' such as a protein. Knowing the three-dimensional structure of the active part can enable scientists to design a synthetic analogue of the active part that will block, mimic or enhance the natural biological activity of the molecule. (Appelt, K. et al.,
J. Med. Chem.,
34:1925 (1991)). The biological molecule may, for example, be a receptor, an enzyme, a hormone, or other biologically active molecule. Determining the three-dimensional structures of biological molecules is, therefore, of great practical and commercial significance.
The first technique developed to determine three-dimensional structures was X-ray crystallography. The structures of hemoglobin and DNA were determined using this technique. In X-ray crystallography, a crystal (or fiber) of the material to be examined is bombarded with a beam of X-rays which are refracted by the atoms of the ordered molecules in the crystal. The scattered X-rays are captured on a photographic plate which is then developed using standard techniques. The diffracted X-rays are thus visualized as a series of spots on the plate and from this pattern, the structure of the molecules in the crystal can be determined. For larger molecules, it is frequently necessary to crystallize the material with a heavy ion, such as ruthenium, in order to remove ambiguity due to phase differences.
More recently, a second technique, nuclear magnetic resonance (NMR) spectroscopy, has been developed to determine the three-dimensional structures of biological molecules, particularly proteins. NMR was originally developed in the 1950's and has evolved into a powerful procedure to analyze the structure of small compounds such as those with a molecular weight of ≦1000 Daltons. Briefly, the technique involves placing the material to be examined (usually in a suitable solvent) in a powerful magnetic field and irradiating it with radio frequency (rf) electromagnetic radiation. The nuclei of the various atoms will align themselves with the magnetic field until energized by the rf radiation. They then absorb this resonant energy and re-radiate it at a frequency dependent on i) the type of nucleus and ii) its atomic environment. Moreover, resonant energy can be passed from one nucleus to another, either through bonds or through three-dimensional space, thus giving information about the environment of a particular nucleus and nuclei in its vicinity.
However, it is important to recognize that not all nuclei are NMR active. Indeed, not all isotopes of the same element are active. For example, whereas “ordinary” hydrogen,
1
H, is NMR active, heavy hydrogen (deuterium),
2
H, is not active in the same way. Thus, any material that normally contains
1
H hydrogen can be rendered “invisible” in the hydrogen NMR spectrum by replacing all the
1
H hydrogens with
2
H. It is for this reason that NMR spectroscopic analyses of water-soluble materials frequently are performed in
2
H
2
O to eliminate the water signal.
Conversely, “ordinary” carbon,
12
C, is NMR inactive whereas the stable isotope,
13
C, present to about 1% of total carbon in nature, is active. Similarly, while “ordinary” nitrogen,
14
N, is nmr active, it has undesirable properties for NMR and resonates at a different frequency from the stable isotope
15
N, present to about 0.4% of total nitrogen in nature. For small molecules, these low level natural abundances were sufficient to generate the required experimental information, provided that the experiment was conducted with sufficient quantities of material and for a is sufficient time.
As advances in hardware and software were made, the size of molecules that could be analyzed by these techniques increased to about 10 kD, the size of a small protein. Thus, the application of NMR spectroscopy to protein structural determinations began only a few years ago. It was quickly realized that this size limit could be raised by substituting the NMR inactive isotopes
14
N and
12
C in the protein with the NMR active stable isotopes
15
N and
13
C.
Over the past few years, labeling proteins with
15
N and
15
N/
13
C has raised the analytical molecular size limit to approximately 15 kD and 40 kD, respectively. More recently, partial deuteration of the protein in addition to
13
C- and
15
N-labeling has increased the size of proteins and protein complexes still further, to approximately 60-70 kD. See Shan et al.,
J. Am. Chem.Soc.,
118:6570-6579 (1996) and references cited therein.
Isotopic substitution is usually accomplished by growing a bacterium or yeast, transformed by genetic engineering to produce the protein of choice, in a growth medium containing
13
C-,
15
N- and/or
2
H-labeled substrates. In practice, bacterial growth media usually consist of
13
C-labeled glucose and/or
15
N-labeled ammonium salts dissolved in D
2
O where necessary. Kay, L. et al.,
Science,
249:411 (1990) and references therein and Bax, A.,
J. Am. Chem. Soc.,
115, 4369 (1993). More recently, isotopically labeled media especially adapted for the labeling of bacterially produced macromolecules have been described. See U.S. Pat. No. 5,324,658.
The goal of these methods has been to achieve universal and/or random isotopic enrichment of all of the amino acids of the protein. By contrast, some workers have described methods whereby certain residues can be relatively enriched in
1
H,
2
H,
13
C and
15
N. For example, Kay et al.,
J. Mol. Biol.,
263, 627-636 (1996) and Kay et al.,
J. Am. Chem. Soc.,
119, 7599-7600 (1997) have described methods whereby isoleucine, alanine, valine and leucine residues in a protein may be labeled with
2
H,
13
C and
15
N, but specifically labeled with
1
H at the terminal methyl position. In this way, study of the proton-proton interactions between some of the hydrophobic amino acids may be facilitated. Similarly, a cell-free system has been described by Yokoyama et al.,
J. Biomol. NMR,
6(2), 129-134 (1995)., wherein a transcription-translation system derived from
E. coli
was used to express human Ha-Ras protein incorporating
15
N serine and/or aspartic acid.
These methods are important, in that they provide additional means for interpreting the complex spectra obtained from proteins. However, it should be noted that the Kay et al. methods are limited to the aliphatic amino acids described above. By contrast, the method described by Yokoyama will facilitate the selective enrichment of any amino acid, but is limited to those proteins that can be expressed in a cell-free system. Glycoproteins, for example, may not be expressed in this system.
Techniques for producing isotopically labeled proteins and macromolecules, such as glycop

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