Proteins: Structure, Function, and

A prior distribution for sequence profiles can be derived from mixtures of Dirichlet distributions [16,51-54]. The idea is simple Each position in a multiple alignment represents one of a limited number of possible distributions that reflect the important physical forces that determine protein structure and function. In certain core positions, we expect to get a distribution restricted to Val, He, Met, and Leu. Other core positions may include these amino acids plus the large hydrophobic aromatic amino acids Phe and Trp. There will also be positions that are completely conserved, including catalytic residues (often Lys, GIu, Asp, Arg, Ser, and other polar amino acids) and Gly and Pro residues that are important in achieving certain backbone conformations in coil regions. Cys residues that form disulfide bonds or coordinate metal ions are also usually well conserved. 

Special Focus Hemoglobin and Myoglobin— Paradigms of Protein Structure and Function... 

Harding SE, Jumel K, Kelly R, Gudo E, Horton JC, Mitchell JR (1993) In Schwenke KD, Mothes R (eds) Food Proteins Structure and Functionality. VCH, Weinheim, Germany, 216... 

In most cases, biological activity of proteins is dependent on specific three-dimensional (3D) tertiary strucmres. Not surprisingly, as a consequence of this interest in protein structure and function. 

Proteins derive their powerful and diverse capacity for molecular recognition and catalysis from their ability to fold into defined secondary and tertiary structures and display specific functional groups at precise locations in space. Functional protein domains are typically 50-200 residues in length and utilize a specific sequence of side chains to encode folded structures that have a compact hydrophobic core and a hydrophilic surface. Mimicry of protein structure and function by non-natural ohgomers such as peptoids wiU not only require the synthesis of >50mers with a variety of side chains, but wiU also require these non-natural sequences to adopt, in water, tertiary structures that are rich in secondary structure. 

If cellular redox state, determined by the glutathione status of the heart, plays a role in the modulation of ion transporter activity in cardiac tissue, it is important to identify possible mechanisms by which these effects are mediated. Protein S-,thiolation is a process that was originally used to describe the formation of adducts of proteins with low molecular thiols such as glutathione (Miller etal., 1990). In view of the significant alterations of cardiac glutathione status (GSH and GSSG) and ion-transporter activity during oxidant stress, the process of S-thiolation may be responsible for modifications of protein structure and function. 

Figure 4.14 Diagrammatic representation of (a) oxy-radical>mediated S-thioiation and (b) thiol/disulphide-initiated S-thiolation of protein suiphydryl groups. Under both circumstances mixed disuiphides are formed between glutathione and protein thiois iocated on the ion-translocator protein resulting in an alteration of protein structure and function. Both of these mechanisms are completely reversible by the addition of a suitabie reducing agent, such as reduced glutathione, returning the protein to its native form.

Scheraga, H. A., Protein structure and function, from a colloidal to a molecular view, Carlsberg Res. Comm., 49, 1, 1984. 

Spudich, J. L., C. S. Yang et al. (2000). Retinylidene proteins Structures and functions from archaea to humans. Annu. Rev. Cell Dev. Biol. 16 365-392. 

Horovitz, A., Double-mutant cycles a powerful tool for analyzing protein structure and function, Fold. Des. 1996,1, R121-126. 

Sklonick J et al. From genes to protein structure and function novel applications of computational approaches in the genomic era. TIBTECH 2000 18 34-39. 

Kelly SM, Price NC. The use of circular dichroism in the investigation of protein structure and function. Curr. Protein Pept. Sci. 2000 1 349-384. 

It is obvious that the oxidation of protein molecules can have detrimental effects on protein structure and function. However, there are some unique methods in bioconjugation wherein controlled and purposeful oxidation is done to study protein-protein interactions (Chapter 28, Section 4). 

Many of the initial biopharmaceuticals approved were simple replacement proteins (e.g. blood factors and human insulin). The ability to alter the amino acid sequence of a protein logically coupled to an increased understanding of the relationship between protein structure and function (Chapters 2 and 3) has facilitated the more recent introduction of several engineered therapeutic proteins (Table 1.3). Thus far, the vast majority of approved recombinant proteins have been produced in the bacterium E. coli, the yeast S. cerevisiae or in animal cell lines (most notably Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells. These production systems are discussed in Chapter 5. 

Ras and its relatives are subjects of intensive investigations by biological, biochemical, biophysical, and medical studies. Within just one decade more than 17,000 articles (Medline, 1966-2000) deal with function and properties of this protein. Structural and functional data, based on Ras as a prototype, have provided insight into the basic principles of GTP-binding proteins, their activation, de-activation, and signal transmission. 

Clearly the relationships between protein structure and function and inherent sensitizing activity are complicated and far from fully defined, and it is against this background of uncertainty that it is necessary to develop approaches suitable for hazard identification. 

Protein domains are the common currency of protein structure and function. Protein domains are discrete structural units that fold up to form a compact globular shape. Experiments on protein structure and function have been greatly aided by consideration of the modular nature of proteins. This has allowed very large proteins to be studied. The expression of individual domains has allowed the intractable giant muscle protein titin to be structurally studied (Pfuhl and Pastore, 1995). Protein domains can be found in a variety of contexts, (Fig. 1), in association with a range of unrelated domains and in a variety of orders. Ultimately protein domains are defined at the level of three-dimensional structure however, many protein domains have been described at the level of sequence. The success of sequence-based methods has been demonstrated by numerous confirmations, by elucidation of the three-dimensional structure of the domain. 

Prior to the advent of site-directed mutagenesis as a viable technique for the production of specifically modified proteins, the last major event to exert a major influence on the study of protein structure and function was the development of X-ray diffraction analysis for the detailed structural analysis of macromolecules. In the intervening thirty years, the availability of protein structures obtained in this manner combined with a wide range of physical and chemical studies of these proteins allowed development of substantial insight into the relationship between the structure of a protein and its functional attributes. There was some reason to expect, therefore, that functional characterization of specifically mutated proteins based on understanding developed with more classical techniques should permit efficient confirmation of existing hypotheses, particularly for proteins for which the available literature is as extensive as that for cytochrome c. 

Huibregtse, J. M., et al., A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc Natl Acad Sci USA, 1995, 92(7), 2563-7. 

Chimeragenesis and SDM are powerful techniques that can be used to investigate the complex relationships between protein structure and function. The methods detailed here are relatively simple to perform and can be carried out in a short period of time. They are applicable to any protein type for which the cDNA is available and can be modified for many different purposes in protein engineering. 

Posttranslational modifications are a sundry set of transformations that help to diversify the limited genome of organisms. The modifications discussed in this chapter have been shown to modify a wide variety of proteins whose functions vary from cell division to metabolism and regulation. While a large selection of posttranslational modifications has been discussed, the presentation is not all-inclusive of all modifications. Emphasis has been placed on the discoveries made since 2005 and on the more common modifications. The importance of posttranslational modifications on protein structure and function and cellular function has been emphasized. 

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