Protein sequence-structure sample structures

A related application for RDCs has also been described based on the sequence-dependent pattern of RDCs along a helical structure, called a dipolar wave by the Opella group [317, 352, 353]. The magnitude and periodicity of the dipolar wave depends on the orientation of the helix, and can allow irregularities in helix structure to be identified. Most commonly, dipolar waves have been used to help determine the location of helices in a protein sequence, allowing these structural elements to be more rigidly restrained over the course of a structure calculation [57,159, 323, 354]. This is particularly useful for larger helical membrane proteins, since a-helices are not well defined by the NOEs available in sparsely protonated samples [262]. 

The amount of sample required is quite small as little as 10 mole is typical So many peptides and proteins have been sequenced now that it is impossible to give an accurate count What was Nobel Prize winning work m 1958 is routine today Nor has the story ended Sequencing of nucleic acids has advanced so dramatically that it is possible to clone the gene that codes for a particular protein sequence its DNA and deduce the structure of the protein from the nucleotide sequence of the DNA We 11 have more to say about DNA sequencing m the next chapter... 

Peptide and protein sequencing and its importance in the proteomics field were discussed in Section 2.2.3. The following gives a brief description of the mass spectrometry methods used to achieve sequencing. First, to produce protein or oligonucleotide structural/sequence information by mass spectro-metric techniques, one needs to use tandem mass spectrometry (MS-MS). In this technique, a sample is first fragmented and analyzed in one mass spec-... 

Macromolecules are very much like the crystalline powder just described. A few polymers, usually biologically-active natural products like enzymes or proteins, have very specific structure, mass, repeat-unit sequence, and conformational architecture. These biopolymers are the exceptions in polymer chemistry, however. Most synthetic polymers or storage biopolymers are collections of molecules with different numbers of repeat units in the molecule. The individual molecules of a polymer sample thus differ in chain length, mass, and size. The molecular weight of a polymer sample is thus a distributed quantity. This variation in molecular weight amongst molecules in a sample has important implications, since, just as in the crystal dimension example, physical and chemical properties of the polymer sample depend on different measures of the molecular weight distribution. 

Each protein in a sample is unique and can demonstrate that individuality in protein assays as variation in the color response. Such protein-to-protein variation refers to differences in the amount of color (absorbance) that are obtained when the same mass (microgram or milligram) of various proteins are assayed concurrently (i.e., in the same run) by the same method. These differences in color response relate to differences among proteins due to amino acid sequence, isoelectric point (pi), secondary structure, and the presence of certain side chains or prosthetic groups. 

The mass spectrometry does not involve an interaction between electromagnetic radiation and sample molecule. The functions of a mass spectrometer are to produce positive ions from the sample under investigation, to resolve these ions into a series of ion beams that are homogeneous with respect to their mass/charge ratio (m/e), and to measure the relative abundance of the ions in these beams. The main applications include the molecular weight and structural determinations (Bieman, 1992). Mass spectrometry has emerged as the method for rapid analyses of protein sequences and annotation of their databases (Mann and Pandey, 2001). 

Automated carboxy-terminal (C-terminal) protein sequence analysis enables the direct and unambiguous confirmation of the C-terminal sequence of native and expressed proteins, the detection and characterization of protein processing at the C-terminus, the identification of post-translational proteolytic cleavages, and partial sequence information on amino-terminally blocked protein samples. In order for C-terminal sequence analysis to be of immediate benefit, each of the 20 common amino acid residues must be detectable. Additionally, the scope of typically analyzable protein samples must span a usefully broad molecular weight range and degree of structural complexity. 

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