H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 29
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In addition, the cell has error-checking processes thateliminate incorrectly synthesized or folded proteins. Incorrectly folded proteins usually lack biological activity and, insome cases, may actually be associated with disease. Proteinmisfolding is suppressed by two distinct mechanisms. First,cells have systems that reduce the chances for misfolded proteins to form.
Second, any misfolded proteins that do form,as well as cytosolic proteins no longer needed by a cell, are degraded by a specialized cellular garbage-disposal system.The Information for Protein Folding Is Encodedin the SequenceAny polypeptide chain containing n residues could, in principle, fold into 8n conformations. This value is based on thefact that only eight bond angles are stereochemically allowedin the polypeptide backbone.
In general, however, all molecules of any protein species adopt a single conformation,called the native state; for the vast majority of proteins, thenative state is the most stably folded form of the molecule.What guides proteins to their native folded state? The answer to this question initially came from in vitro studies onprotein refolding. Thermal energy from heat, extremes of pHthat alter the charges on amino acid side chains, and chemicals such as urea or guanidine hydrochloride at concentrations of 6–8 M can disrupt the weak noncovalent interactionsthat stabilize the native conformation of a protein. Thedenaturation resulting from such treatment causes a proteinto lose both its native conformation and its biological activity.Many proteins that are completely unfolded in 8 M ureaand -mercaptoethanol (which reduces disulfide bonds) spontaneously renature (refold) into their native states when the denaturing reagents are removed by dialysis.
Because no cofactors3.2 • Folding, Modification, and Degradation of Proteins69class of proteins found in all organisms from bacteria to humans. Chaperones are located in every cellular compartment,bind a wide range of proteins, and function in the generalprotein-folding mechanism of cells. Two general families ofchaperones are reconized:or other proteins are required, in vitro protein folding is a selfdirected process. In other words, sufficient information mustbe contained in the protein’s primary sequence to direct correct refolding. The observed similarity in the folded, threedimensional structures of proteins with similar amino acidsequences, noted in Section 3.1, provided other evidence thatthe primary sequence also determines protein folding in vivo.■ Molecular chaperones, which bind and stabilize unfolded or partly folded proteins, thereby preventing theseproteins from aggregating and being degradedFolding of Proteins in Vivo Is Promotedby Chaperones■Although protein folding occurs in vitro, only a minority ofunfolded molecules undergo complete folding into the nativeconformation within a few minutes.
Clearly, cells require afaster, more efficient mechanism for folding proteins intotheir correct shapes; otherwise, cells would waste much energy in the synthesis of nonfunctional proteins and in thedegradation of misfolded or unfolded proteins. Indeed, morethan 95 percent of the proteins present within cells have beenshown to be in their native conformation, despite high protein concentrations (200–300 mg/ml), which favor the precipitation of proteins in vitro.The explanation for the cell’s remarkable efficiency inpromoting protein folding probably lies in chaperones, aMolecular chaperones consist of Hsp70 and its homologs:Hsp70 in the cytosol and mitochondrial matrix, BiP in the endoplasmic reticulum, and DnaK in bacteria.
First identifiedby their rapid appearance after a cell has been stressed by heatshock, Hsp70 and its homologs are the major chaperones inall organisms. (Hsc70 is a constitutively expressed homolog ofHsp70.) When bound to ATP, Hsp70-like proteins assume anopen form in which an exposed hydrophobic pocket transiently binds to exposed hydrophobic regions of the unfoldedtarget protein. Hydrolysis of the bound ATP causes molecular chaperones to assume a closed form in which a target protein can undergo folding. The exchange of ATP for ADPreleases the target protein (Figure 3-11a, top).
This cycle isChaperonins, which directly facilitate the folding ofproteins(a)(b)RibosomeProteinPartiallyfoldedproteinATPProperlyfoldedproteinGroEL "tight"conformationADP+PiProteinProperlyfoldedproteinATPGroESGroEL▲ FIGURE 3-11 Chaperone- and chaperonin-mediatedprotein folding. (a) Many proteins fold into their proper threedimensional structures with the assistance of Hsp70-like proteins(top). These molecular chaperones transiently bind to a nascentpolypeptide as it emerges from a ribosome.
Proper folding ofother proteins (bottom) depends on chaperonins such as theprokaryotic GroEL, a hollow, barrel-shaped complex of 14identical 60,000-MW subunits arranged in two stacked rings.GroEL "relaxed"conformationOne end of GroEL is transiently blocked by the cochaperonin GroES, an assembly of 10,000-MW subunits.(b) In the absence of ATP or presence of ADP, GroEL existsin a “tight” conformational state that binds partly folded ormisfolded proteins.
Binding of ATP shifts GroEL to a moreopen, “relaxed” state, which releases the folded protein.See text for details. [Part (b) from A. Roseman et al., 1996, Cell87:241; courtesy of H. Saibil.]MEDIA CONNECTIONSHsp 70-ATPADPFocus Animation: Chaperone-Mediated FoldingPi70CHAPTER 3 • Protein Structure and Functionspeeded by the co-chaperone Hsp40 in eukaryotes. In bacteria,an additional protein called GrpE also interacts with DnaK,promoting the exchange of ATP for the bacterial co-chaperoneDnaJ and possibly its dissociation. Molecular chaperones arethought to bind all nascent polypeptide chains as they arebeing synthesized on ribosomes.
In bacteria, 85 percent of theproteins are released from their chaperones and proceed tofold normally; an even higher percentage of proteins in eukaryotes follow this pathway.The proper folding of a large variety of newly synthesizedor translocated proteins also requires the assistance of chaperonins. These huge cylindrical macromolecular assemblies areformed from two rings of oligomers. The eukaryotic chaperonin TriC consists of eight subunits per ring.
In the bacterial,mitochondrial, and chloroplast chaperonin, known as GroEL,each ring contains seven identical subunits (Figure 3-11b). TheGroEL folding mechanism, which is better understood thanTriC-mediated folding, serves as a general model (Figure3-11a, bottom). In bacteria, a partly folded or misfoldedpolypeptide is inserted into the cavity of GroEL, where it bindsto the inner wall and folds into its native conformation. In anATP-dependent step, GroEL undergoes a conformationalchange and releases the folded protein, a process assisted by aco-chaperonin, GroES, which caps the ends of GroEL.Many Proteins Undergo Chemical Modificationof Amino Acid ResiduesNearly every protein in a cell is chemically modified after itssynthesis on a ribosome.
Such modifications, which mayalter the activity, life span, or cellular location of proteins,entail the linkage of a chemical group to the free –NH2 or–COOH group at either end of a protein or to a reactive sidechain group in an internal residue. Although cells use the 20amino acids shown in Figure 2-13 to synthesize proteins,analysis of cellular proteins reveals that they contain upwardof 100 different amino acids. Chemical modifications aftersynthesis account for this difference.Acetylation, the addition of an acetyl group (CH3CO) tothe amino group of the N-terminal residue, is the most common form of chemical modification, affecting an estimated80 percent of all proteins:RONCCHHOCH3COAcetyl lysineCH3CNCH2CH2CH2COOCHCH2NH3OPhosphoserine−OPCH2OCHCOONH3O−OH3-HydroxyprolineH2CCHH2CCHHC3-MethylhistidineH 3CNCH2CHCOONH3NOOC -CarboxyglutamateCHNH2CCHCOOOOCCH2CHCOONH3▲ FIGURE 3-12 Common modifications of internal aminoacid residues found in proteins.
These modified residues andnumerous others are formed by addition of various chemicalgroups (red) to the amino acid side chains after synthesis of apolypeptide chain.Acetyl groups and a variety of other chemical groups canalso be added to specific internal residues in proteins (Figure 3-12). An important modification is the phosphorylationof serine, threonine, tyrosine, and histidine residues. We willencounter numerous examples of proteins whose activity isregulated by reversible phosphorylation and dephosphorylation. The side chains of asparagine, serine, and threonineare sites for glycosylation, the attachment of linear andbranched carbohydrate chains. Many secreted proteins andmembrane proteins contain glycosylated residues; the synthesis of such proteins is described in Chapters 16 and 17.Other post-translational modifications found in selected proteins include the hydroxylation of proline and lysine residuesin collagen, the methylation of histidine residues in membrane receptors, and the -carboxylation of glutamate inprothrombin, an essential blood-clotting factor.
A specialmodification, discussed shortly, marks cytosolic proteins fordegradation.Acetylated N-terminusThis modification may play an important role in controllingthe life span of proteins within cells because nonacetylatedproteins are rapidly degraded by intracellular proteases.Residues at or near the termini of some membrane proteins arechemically modified by the addition of long lipidlike groups.The attachment of these hydrophobic “tails,” which functionto anchor proteins to the lipid bilayer, constitutes one way thatcells localize certain proteins to membranes (Chapter 5).Peptide Segments of Some Proteins Are RemovedAfter SynthesisAfter their synthesis, some proteins undergo irreversiblechanges that do not entail changes in individual amino acidresidues.
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