Higher order protein structure

Despite the multiple challenges faced by the top-down HX-MS, the outlook for this technique remains very bright. It has already made tremendous progress in the past decade, effectively transitioning from an esoteric curiosity in the field of experimental biophysics with very few followers to a powerful tool uniquely capable of solving complex problems related to protein higher-order structure and dynamics. It is clear that further developments in the top-down HX-MS will continue to expand the boundaries of this technique and eventually transform it into a routine tool in the experimental armamentarium of structural biology and biophysics. 

Griffith, W.P., Mohimen, A., Abzalimov, R.R., Kaltashov, I.A. (2008) Characterization of protein higher order structure and dynamics with ESI MS, in Mass Spectrometry of Proteins (ed. J.P. Whitelegge),... 

Figure 3 Examples of higher-order structure in proteins the a-helix and P-sheet.

The essential distinction between the approaches used to formulate and evaluate proteins, compared with conventional low molecular weight drugs, lies in the need to maintain several levels of protein structure and the unique chemical and physical properties that these higher-order structures convey. Proteins are condensation polymers of amino acids, joined by peptide bonds. The levels of protein architecture are typically described in terms of the four orders of structure [23,24] depicted in Fig. 2. The primary structure refers to the sequence of amino acids and the location of any disulfide bonds. Secondary structure is derived from the steric relations of amino acid residues that are close to one another. The alpha-helix and beta-pleated sheet are examples of periodic secondary structure. Tertiary... 

Protrusion may be due to growth of new actin filaments, which requires net polymerisation of new filaments, and also by the organisation of actin-binding proteins into higher-order structures. Random movements of flexible membranes away from the filaments may result in gross distortion of actin polymerisation at the barbed ends. Thus, once a critical size is reached, ion pumping (i.e. of Ca2+) may occur at the tip of a pseudopod, which further aids directional changes in the network. 

Since 1974, evidence has accumulated in the literature which indicates that chromatin itself may be considered as an assembly system. It is true that chromatin is more complex than assembly systems analyzed to date, both with respect to the size of the nucleic acid involved and therefore the amount (and variety) of protein complexed with it and with respect to the dynamic aspect of the multilevel higher order structure. Nevertheless, at least at the lower levels of organization, the interpretation of chromatin as an assembly system may be valid. Evidence for this derives from three basic lines of research described in previous sections (1) the reconstitution of the nucleosome, (2) the self-assembly of the octamer, and (3) the putative self-organization of nucleosomes into higher order structures. 

However, it is still possible that other mechanisms may exist to bridge the gap between substrate and E2 in the SCF-mediated ubiquitin-transfer reaction. For example, reports suggest that SCF may form higher order structures to facilitate the degradation of protein substrates. The S. pombe F-box proteins Popl and Pop2 have been shown to form heterodimers, and evidence suggests that these interactions may be important for the degradation of their in vivo substrates [62]. 

To say that RNA molecules are single-stranded molecules is not the same as saying that they have no higher-order structures, hi fact they have several. The formation of Watson-Crick complementary base pairs is a driving force for formation of higher-order structures. These include the stem-loop and hairpin secondary structures, as well as more complex tertiary structures. Of particular note, are the complex structures for transfer RNAs, tRNAs. Examples are provided in figure 12.5 (note that there are several nnnsnal bases in these structnres this is typical of tRNAs but not of RNA molecnles in general). These strnctures are intimately related to the function of these molecnles as adaptors in the process of protein synthesis, as developed in the next chapter. 

Of the five snRNAs, U2 and U6 interact with the reaction site (the 5 splice site and the branch point) in the first chemical step. These two snRNAs are known to anneal together to form a stable-based paired structure in the absence of proteins and in the presence of ions as shown in Fig. 13, with U2 acting as an inducer molecule that displaces the U4 (that is an antisense molecule that regulates the catalytic function of U6 RNA) from the initially formed U4-U6 duplex. The secondary (or higher ordered) structure of the U2-U6 complex consists of the active site of the spliceosome. Recent data suggests that these two snRNAs function as the catalytic domain of the spliceosome that catalyzes the first step of the splicing reaction [145]. 

Histones are very basic proteins with an isoelectric point between 10.31 and 11.27 for human complement. They are present in virtually all eukaryotes (with the exception of dinoflagellates [14]) where they are associated with most of the nuclear DNA. The DNA is wrapped around an octamer formed by the four core histones H2A, H2B, H3 and H4 to build a nucleosome. This particle is the fundamental repeating unit of chromatin [15]. A string of nudeosomes can fold into a higher order structure, the exact molecular nature of which is still not fully understood but clearly has a strong influence on gene expression. 

Like proteins, DNA and RNA exhibit higher order structure that dictates how these molecules function. Determining the structure of DNA challenged the world s foremost scientists during the middle of the... 

At the cytological level, the power and resolution of a variety of chromosome staining and banding techniques has been increased by ihcir application to prophase chromosomes and the genetic map now locates over one thousand bands. At the nuclcosomal level, the association of DNA with histone proteins is reasonably well understood. However, knowledge of the higher order structure and the nature of the association between DNA and the acidic structural scaffold, or core proteins, of the chromosome, remains unresolved. 

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