Small and Medium-Sized Proteins

The first experiment to be recorded on isotope-labeled proteins is the [ N/HJ-HSQC experiment. Inspection of the [ N. HJ-correlalion map and simple counting of cross peaks reveals whether multiple conformers exist, whether some parts of the backbone signals are broadened, possibly because of slow conformational exchange, or whether parts of the sequence are not visible at all. As mentioned above, the spectrum will also show whether the protein is well folded or not. 

The larger the protein, the greater the resonance overlap. Often several different residues have degenerate 13Ca frequencies which will make an unambiguous assignment difficult or even impossible. In these cases, additional information from different NMR experiments is required. One possibility is to use the carbonyl chemical shift instead of the Ca chemical shift and measure the HNCO/HN(CA)CO pulse sequence pair [37, 45, 46, 49, 50]. As with the HNCA/HN(CO)CA combination, one of the experiments, the 

In the CBCA(CO)NH experiment the coherence pathway includes the carbonyl spin, which suppresses the intra-residual coherence transfer. However, as for the other two pairs of pulse sequences mentioned above, an experiment can be designed that allows simultaneous intra- and inter-residual coherence transfer. In the CBCANH experiment 

Once the sequential assignment is complete, the secondary structure of the protein can be determined from the secondary-structure specific chemical shifts of the 13Ca nuclei. Other nuclei, including the 13C/9 and the 13CO, also show characteristic chemical shifts that can be used to identify the secondary structure [55-60]. In addition, the secondary structure can also be assigned with the help of the identified secondary structure-specific sequential NOEs and coupling constants obtained from the HNHA experiment 

A more sensitive alternative is to use HCCH-TOCSY experiments [63-65], which transfer magnetization through the much larger 13C-13C coupling constants. In addition, these coupling constants are almost independent of the secondary structure and very uniform 

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Two methods for the measurement of /NCaipha and /NCaipha in N Relabeled small and medium-size proteins are described by Heikkinen and co-... 

In this book, we concentrate largely on methods for the computer manipulation of small and medium-sized molecules, molecules of up to a few hundred or thousand atoms. We do this to develop an understanding of the methods available for the processing of information on chemical compounds and reactions. However, many of these methods can also be applied to macromolecules such as proteins and nucleic acids. 

Blood is the transport medium of the body. Plasma, which accounts for approximately 60% of the total volume, carries a wide range of small and medium-sized metabolites some are simply dissolved in solution (93% of the plasma is water), others are carried by specific carrier proteins. The chemical composition of the plasma is complex and reflects the chemical composition inside cells, which is why blood tests are so commonly used in diagnosis to see the biochemical events occurring in tissues. The formed cellular elements of the blood perform several functions defence against blood loss from bleeding (platelets, also called thrombocytes), defence against infection and immune surveillance (white cells, leucocytes), and gas transport and pH buffering (red cells, erythrocytes). 

Direct methods work if the molecules, and thus the unit cells and numbers of reflections, are relatively small. Isomorphous replacement works if the molecules are large enough that a heavy atom does not disturb their structures significantly. The most difficult structures for crystallographers are those that are too large for direct methods and too small to remain isomorphous despite the intrusion of a heavy atom. If a medium-size protein naturally contains a heavy atom, like iron or zinc, or if a selenomethionine derivative can be produced, the structure can often be solved by MAD phasing (Section IV.E). [NMR methods (see Chapter 10) are also of great power for small and medium-size molecules ]... 

PKR1 and PKR2 mRNAs are expressed in DRG of neonatal and adult rats PKR1 is mainly expressed in small and medium size neurons and PKR2 in large neurons. PKR proteins are present in DRG, in the outer layers of the dorsal horns of the spinal cord and in peripheral terminals of nociceptor axons (Negri et at, 2006a,b). 

A simple approach to protein description consists of representing a protein by a sequence of properties of its constituent amino acids. Each amino acid is described by one ore more properties and therefore the total number of protein descriptors is given by the product of the number of amino acids in the protein and the number of selected amino acid properties. As this number of descriptors increases very fast with the size of proteins, this approach is usually applied to small- and medium-size peptides. Moreover, in QSAR studies that require uniform-length descriptors, it can be used only to describe a series of peptide analogues, vhich are peptide sequences with the same length. To enable QSAR studies of peptide sequences with different length, some method is required that is able to translate the peptide sequences into vectorial descriptors with the same number of variables. For example, ACC transforms were applied to compress information about principal properties of amino acids into peptide sequences with different length. 

A relatively small number of biotechnology companies operate in the field of recombinant proteins production in plants (Table 6.4). This small number represents roughly one-third of approximately 60 dedicated plant biotechnology companies - an unfavorable comparison to the several hundred small and medium-sized businesses that use bacterial, yeast or animal-based expression platforms and operate in the sector of red biotechnology. The number of plant-made recombinant proteins that have reached the market is also very limited, but has a healthy tendency to grow. 

A. Small and Medium-Size Globular Proteins Consisting of One Polypeptide Chain... 

According to their chemical structures, CSPs can be divided into three different groups. A multitude of chiral stationary phases is derived from (modified) natural or synthetic polymers, e.g., the polysaccharides, proteins or polyacrylamides. A second type of selectors is based on large chiral ring systems, such as cyclo-dextrins, macTocycKc antibiotics, and crown ethers. The last group comprises moleailes of small and medium size, such as amino acids and their derivatives, alkaloids, and fuUy synthetic selectors. 

Many examples of proteins that have been used in interfecial adsorption studies may be quoted such as small and medium size globular proteins, e.g. those present in milk such as p-lactoglobulin, a-lactoalbumin and serum albumin, and egg white, e.g. lysozyme and ovalalbumin. At pH values below the isoelectric point (4.2-4.S), these proteins associate to form dimers, trimers and higher aggregates, a-lactoalbumin is stabilized by Ca against thermal unfolding. X-ray analysis of lysozyme showed that... 

Currently the availability of a plethora of multi-dimensional/multinuclear NMR methods allows us to extract information on the structures and dynamic behaviours of a wide range of drug molecules of up to 30 kDa in size. These include the small and medium-sized traditionally used therapeutic drugs, to higher molecular weight peptides, proteins, nucleotides, nucleic acids and polysaccharide biotechnology products. 

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