2 Структура и функция белка (1160071), страница 22
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The a helices line a long crevice in the side ofthe molecule (Fig. 7-20), called the active site, which is the site ofsubstrate binding and action. The bacterial polysaccharide that is thesubstrate for lysozyme fits into this crevice.Ribonuclease, another small globular protein (Mr 13,700), is anenzyme secreted by the pancreas into the small intestine, where itcatalyzes the hydrolysis of certain bonds in the ribonucleic acids present in ingested food. Its tertiary structure, determined by x-ray analysis, shows that little of its 124 amino acid polypeptide chain isin a-helical conformation, but it contains many segments in the/3 conformation.
Like lysozyme, ribonuclease has four disulfide bondsbetween loops of the polypeptide chain (Fig. 7-20).Table 7-2 shows the relative percentages of a helix and /3 conformation among several small, single-chain, globular proteins. Each ofthese proteins has a distinct structure, adapted for its particular biological function. These proteins do share several important properties,however. Each is folded compactly, and in each case the hydrophobicamino acid side chains are oriented toward the interior (away fromwater) and the hydrophilic side chains are on the surface. These specific structures are also stabilized by a multitude of hydrogen bondsand some ionic interactions.Proteins Lose Structure and Function on DenaturationThe way to demonstrate the importance of a specific protein structurefor biological function is to alter the structure and determine the effecton function.
One extreme alteration is the total loss or randomizationof three-dimensional structure, a process called denaturation. This isthe familiar process that occurs when an egg is cooked. The white ofthe egg, which contains the soluble protein egg albumin, coagulates toa white solid on heating. It will not redissolve on cooling to yield a clearsolution of protein as in the original unheated egg white. Heating ofegg albumin has therefore changed it, seemingly in an irreversiblemanner.
This effect of heat occurs with virtually all globular proteins,regardless of their size or biological function, although the precise temperature at which it occurs may vary and it is not always irreversible.The change in structure brought about by denaturation is almost invariably associated with loss of function.
This is an expected consequence of the principle that the specific three-dimensional structure ofa protein is critical to its function.Proteins can be denatured not only by heat, but also by extremes ofpH, by certain miscible organic solvents such as alcohol or acetone, bycertain solutes such as urea, or by exposure of the protein to detergents. Each of these denaturing agents represents a relatively mildtreatment in the sense that no covalent bonds in the polypeptide chainare broken. Boiling a protein solution disrupts a variety of weak interactions.
Organic solvents, urea, and detergents act primarily by disrupting the hydrophobic interactions that make up the stable core ofglobular proteins; extremes of pH alter the net charge on the protein,Chapter 7 The Three-Dimensional Structure of Proteins181causing electrostatic repulsion and disruption of some hydrogen bonding. Remember that the native structure of most proteins is only marginally stable. It is not necessary to disrupt all of the stabilizing weakinteractions to reduce the thermodynamic stability to a level that isinsufficient to keep the protein conformation intact.Amino Acid Sequence Determines Tertiary StructureThe most important proof that the tertiary structure of a globular protein is determined by its amino acid sequence came from experimentsshowing that denaturation of some proteins is reversible.
Some globular proteins denatured by heat, extremes of pH, or denaturing reagentswill regain their native structure and their biological activity, a process called renaturation, if they are returned to conditions in whichthe native conformation is stable.A classic example is the denaturation and renaturation of ribonuclease. Purified ribonuclease can be completely denatured by exposureto a concentrated urea solution in the presence of a reducing agent.The reducing agent cleaves the four disulfide bonds to yield eight Cysresidues, and the urea disrupts the stabilizing hydrophobic interactions, thus freeing the entire polypeptide from its folded conformation.Under these conditions the enzyme loses its catalytic activity and undergoes complete unfolding to a randomly coiled form (Fig. 7-21).When the urea and the reducing agent are removed, the randomlycoiled, denatured ribonuclease spontaneously refolds into its correcttertiary structure, with full restoration of its catalytic activity (Fig.7-21).
The refolding of ribonuclease is so accurate that the four intrachain disulfide bonds are reformed in the same positions in the renatured molecule as in the native ribonuclease. In theory, the eight Cysresidues could have recombined at random to form up to four disulfidebonds in 105 different ways. This classic experiment, carried out byChristian Anfinsen in the 1950s, proves that the amino acid sequenceof the polypeptide chain of proteins contains all the information required to fold the chain into its native, three-dimensional structure.The study of homologous proteins has strengthened this conclusion. We have seen that in a series of homologous proteins, such ascytochrome c, from different species, the amino acid residues at certainpositions in the sequence are invariant, whereas at other positions theamino acids may vary (see Fig. 6-15).
This is also true for myoglobinsisolated from different species of whales, from the seal, and from someterrestrial vertebrates. The similarity of the tertiary structures andamino acid sequences of myoglobins from different sources led to theconclusion that the amino acid sequence of myoglobin somehow mustdetermine its three-dimensional folding pattern, an idea substantiatedby the similar structures found by x-ray analysis of myoglobins fromdifferent species. Other sets of homologous proteins also show this relationship; in each case there are sequence homologies as well as similar tertiary structures.Many of the invariant amino acid residues of homologous proteinsappear to occur at critical points along the polypeptide chain.
Some arefound at or near bends in the chain, others at cross-linking points between loops in the tertiary structure, such as Cys residues involved indisulfide bonds. Still others occur at the catalytic sites of enzymes orat the binding sites for prosthetic groups, such as the heme group ofcytochrome c.Native state;catalytically active.Unfolded state;inactive. Disulfidecross-links reduced toyield Cys residues.Native,catalyticallyactive state.Disulfide cross-linksof the four cystineresidues correctlyreformed.Figure 7-21 Renaturation of unfolded, denaturedribonuclease, with reestablishment of correct disulfide cross-links.
Urea is added to denature ribonuclease, and mercaptoethanol (HOCH2CH2SH) to reduce and thus cleave the disulfide bonds of the fourcystine residues to yield eight cysteine residues.182Part II Structure and CatalysisLooking at naturally occurring amino acid substitutions has animportant limitation. Any change that abolishes the function of anessential protein (e.g., a change in an invariant residue) usually results in death of the organism very early in development. This severeform of natural selection eliminates many potentially informativechanges from study.
Fortunately, biochemists have devised methods tospecifically alter amino acid sequences in the laboratory and examinethe effects of these changes on protein structure and function. Thesemethods are derived from recombinant DNA technology (Chapter 28)and rely on altering the genetic material encoding the protein.
By thisprocess, called site-directed mutagenesis, specific amino acid sequences can be changed by deleting, adding, rearranging, or substituting amino acid residues. The catalytic roles of certain amino acids lining the active sites of enzymes such as triose phosphate isomerase andchymotrypsin have been elucidated by substituting different aminoacids in their place.
The importance of certain amino acids in proteinfolding and structure is being addressed in the same way.Tertiary Structures Are Not RigidAlthough the native tertiary conformation of a globular protein is thethermodynamically most stable form its polypeptide chain can assume,this conformation must not be regarded as absolutely rigid.
Globularproteins have a certain amount of flexibility in their backbones andundergo short-range internal fluctuations. Many globular proteins alsoundergo small conformational changes in the course of their biologicalfunction.
In many instances, these changes are associated with thebinding of a ligand. The term ligand in this context refers to a specificmolecule that is bound by a protein (from Latin, ligare, "to tie" or"bind"). For example, the hemoglobin molecule, which we shall examine later in this chapter, has one conformation when oxygen is bound,and another when the oxygen is released. Many enzyme molecules alsoundergo a conformational change on binding their substrates, a process that is part of their catalytic action (Chapter 8).Polypeptides Fold Rapidly by a Stepwise Process(d)Figure 7-22 A possible protein-folding pathway,(a) Protein folding often begins with spontaneousformation of a structural nucleus consisting of afew particularly stable regions of secondary structure, (b) As other regions adopt secondary structure, they are stabilized by long-range interactionswith the structural nucleus, (c) The folding processcontinues until most of the polypeptide has assumed regular secondary structure, (d) The finalstructure generally represents the most thermodynamically stable conformation.In living cells, proteins are made from amino acids at a very high rate.For example, Escherichia coli cells can make a complete, biologicallyactive protein molecule containing 100 amino acid residues in about 5 sat 37 °C.
Yet calculations show that at least 1050 yr would be requiredfor a polypeptide chain of 100 amino acid residues to fold itself spontaneously by a random process in which it tries out all possible conformations around every single bond in its backbone until it finds its native,biologically active form. Thus protein folding cannot be a completelyrandom, trial-and-error process. There simply must be shortcuts.The folding pathway of a large polypeptide chain is unquestionablycomplicated, and the principles that guide this process have not yetbeen worked out in detail. For several proteins, however, there is evidence that folding proceeds through several discrete intermediates,and that some of the earliest steps involve local folding of regions ofsecondary structure. In one model (Fig.
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