Monomeric proteins, folding

To make this chapter as self-contained as possible, we briefly describe lattice models and the commonly employed computational methods. This is followed by a brief description of how a monomeric protein folds. The contents of this section are important to better appreciate the role of chaperones in the rescue of proteins. The chapter is concluded with brief comments about the challenges we face in the straightforward all-atom simulations of protein folding. 

SIMPLIFIED MODELS OF MONOMERIC PROTEIN FOLDING AND DYNAMICS... 

Measuring Protein Sta.bihty, Protein stabihty is usually measured quantitatively as the difference in free energy between the folded and unfolded states of the protein. These states are most commonly measured using spectroscopic techniques, such as circular dichroic spectroscopy, fluorescence (generally tryptophan fluorescence) spectroscopy, nmr spectroscopy, and absorbance spectroscopy (10). For most monomeric proteins, the two-state model of protein folding can be invoked. This model states that under equihbrium conditions, the vast majority of the protein molecules in a solution exist in either the folded (native) or unfolded (denatured) state. Any kinetic intermediates that might exist on the pathway between folded and unfolded states do not accumulate to any significant extent under equihbrium conditions (39). In other words, under any set of solution conditions, at equihbrium the entire population of protein molecules can be accounted for by the mole fraction of denatured protein, and the mole fraction of native protein,, ie. 

Creighton, A. M., Hulford, A., Mant, A., Robinson, D. and Robinson, C. (1995) A monomeric, tightly folded stromal intermediate on the delta pH-dependent thylakoidal protein transport pathway./. Biol. Cbem., 270, 1663-9. 

The van der Waals model of monomeric insulin (1) once again shows the wedge-shaped tertiary structure formed by the two chains together. In the second model (3, bottom), the side chains of polar amino acids are shown in blue, while apolar residues are yellow or pink. This model emphasizes the importance of the hydrophobic effect for protein folding (see p. 74). In insulin as well, most hydrophobic side chains are located on the inside of the molecule, while the hydrophilic residues are located on the surface. Apparently in contradiction to this rule, several apolar side chains (pink) are found on the surface. However, all of these residues are involved in hydrophobic interactions that stabilize the dimeric and hexameric forms of insulin. 

The process through which a linear string of amino acid residues newly synthesized at a ribosome folds into a complex, three-dimensional, biologically active protein structure remains poorly understood. Consider how protein-folding contrasts with RNA-folding. Proteins have 20 distinct monomeric units, RNA only four. The amino acids include aromatic, hydrophobic, cationic, and anionic chemical properties compared to four comparable RNA nucleosides. Moreover, secondary and tertiary structures were fundamentally inter-linked in proteins, but are essentially distinct in RNA molecules. 

The combination of selection (>100-fold enrichment) and screening (1 in 26) shows that fewer than 0.05 % of the possible turn sequences are capable of yielding well-behaved, monomeric proteins. This result again contradicts the simple expectation... 

Cellular and viral IL-lOs and IFN-7 form intercalated dimers (Fig. 2). The intercalated dimer is formed from the first four secondary structural elements from one chain (A-D) and the final two (E and F) from the other. The intertwined dimer fold observed for IFN-7, IL-10, and many other protein structures, is proposed to be an evolutionary mechanism of protein oligomerization referred to as 3D domain swapping (Bennett et at., 1995). This theory suggests IFN-7 and lL-10 evolved from a monomeric protein by exchanging structural domains (a-helices E and E) with another monomer to create the dimer. This hypothesis is strengthened by the discovery of the monomeric IL-lOFMs, lL-19, IL-20, and IL-22. In fact, the close relationship between the intertwined dimeric IFN-7 and monomeric type 1 IFNs was clearly identified when the first structure of IEN-7 was completed in 1991 (Ealick et at, 1991). 

Folding Equilibrium Studies. E. coli RTEM p-lactamase is a monomeric protein. Its amino acid sequence has been determined (79). It has one disulfide bond between the residues Cys S and Cys. The presence of four tyrosines and four tryptophans allows the use of spectroscopic method for the conformational characterization of the enzyme. In this study, the effect of denaturants on the unfolding of p-lactamase was determined from activity measurements, difference spectroscopy and fluorescence intensity measurements. 

Taken together, lipocalins provide attractive candidates in order to engineer novel ligand specificities. Features hke their small size (typically between 150 and 180 residues), monomeric polypeptide composition, dispensable posttranslational modification, and robust protein fold not only facihtate protein-engineering studies but also provide advantages for practical apphcations. 

The stage is now well set for further work addressing more complex questions, such as the study of the folding reaction of oligomers and protein complex formation as well as for studies of aggregation phenomena. Only a few studies have been performed in this direction so far [9-11, 18, 114, 124]. At pressures of 4-8 kbar, most small monomeric proteins unfold... 

Cirilli et al.11171 cloned the gene of diaminopimelate epimerase from Haemophilus influenzae, and purified and crystallized the enzyme. The enzyme is monomeric and has a unique protein fold, in which the amino terminal and carboxyl terminal halves of the molecule fold into structurally homologous and superimposable domains (Fig. 17-13). Cys 73 of the amino terminal domain is found in the disulfide linkage, at the domain interface, with Cys 217 of the carboxy terminal domain 117. Thus, it is most conceivable that these two cysteine residues stay in reduced form in the active enzyme and function as the acid and base in the mechanism. Koo and Blanchard 118 explored a number of kinetic and isotope approaches to clarify the mechanism of the enzyme. However, which of the two cysteine residues is responsible for proton abstraction from the two enantiomeric Ca-H bonds is not yet known. 

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