2 Структура и функция белка (1160071), страница 16
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Orn is ornithine, an amino acid not present in proteins butpresent in some peptides. It has the structureHH3N—CH2—CH2—CH2—C—COO"+NH 3(b) The molecular weight of the peptide was estimated as about 1,200.(c) When treated with the enzyme carboxypeptidase, the peptide failed to undergo hydrolysis.Chapter 6 An Introduction to Proteins(d) Treatment of the intact peptide with 1fluoro-2,4-dinitrobenzene, followed by completehydrolysis and chromatography, yielded only freeamino acids and the following derivative:HO2NNH-CH2-CH2-CH2-C-COO-(Hint: Note that the 2,4-dinitrophenyl derivativeinvolves the amino group of a side chain ratherthan the a-amino group.)159(e) Partial hydrolysis of the peptide followed bychromatographic separation and sequence analysis yielded the di- and tripeptides below (theamino-terminal amino acid is always at the left):Leu-PhePhe-ProOrn-LeuVal-Orn-LeuPhe-Pro-ValVal-OrnPro-Val-OrnGiven the above information, deduce the aminoacid sequence of the peptide antibiotic.
Show yourreasoning. When you have arrived at a structure,go back and demonstrate that it is consistent witheach experimental observation.C H A P T E RThe Three-DimensionalStructure of ProteinsFigure 7-1 The structure of the enzyme chymotrypsin, a globular protein. A molecule of glycine(blue) is shown for size comparison.The covalent backbone of proteins is made up of hundreds of individualbonds. If free rotation were possible around even a fraction of thesebonds, proteins could assume an almost infinite number of threedimensional structures. Each protein has a specific chemical or structural function, however, strongly suggesting that each protein has aunique three-dimensional structure (Fig.
7-1). The simple fact thatproteins can be crystallized provides strong evidence that this is thecase. The ordered arrays of molecules in a crystal can generally formonly if the molecular units making up the crystal are identical. Theenzyme urease (Mr 483,000) was among the first proteins crystallized,by James Sumner in 1926. This accomplishment demonstrated dramatically that even very large proteins are discrete chemical entitieswith unique structures, and it revolutionized thinking about proteins.In this chapter, we will explore the three-dimensional structure ofproteins, emphasizing several principles. First, the three-dimensionalstructure of a protein is determined by its amino acid sequence. Second, the function of a protein depends upon its three-dimensionalstructure.
Third, the three-dimensional structure of a protein isunique, or nearly so. Fourth, the most important forces stabilizing thespecific three-dimensional structure maintained by a given protein arenoncovalent interactions. Finally, even though the structure of proteins is complicated, several common patterns can be recognized.The relationship between the amino acid sequence and the threedimensional structure of a protein is an intricate puzzle that has yet tobe solved in detail.
Polypeptides with very different amino acid sequences sometimes assume similar structures, and similar amino acidsequences sometimes yield very different structures. To find and understand patterns in this biochemical labyrinth requires a renewedappreciation for fundamental principles of chemistry and physics.Overview of Protein StructureThe spatial arrangement of atoms in a protein is called a conformation.
The term conformation refers to a structural state that can, without breaking any covalent bonds, interconvert with other structuralstates. A change in conformation could occur, for example, by rotationabout single bonds. Of the innumerable conformations that are theoretically possible in a protein containing hundreds of single bonds, onegenerally predominates.
This is usually the conformation that is ther160Chapter 7 The Three-Dimensional Structure of Proteins161modynamically the most stable, having the lowest Gibbs' free energy(G). Proteins in their functional conformation are called native proteins.What principles determine the most stable conformation of a protein? Although protein structures can seem hopelessly complex, closeinspection reveals recurring structural patterns.
The patterns involvedifferent levels of structural complexity, and we now turn to a biochemical convention that serves as a framework for much of what follows in this chapter.There Are Four Levels of Architecture in ProteinsConceptually, protein structure can be considered at four levels (Fig.7-2). Primary structure includes all the covalent bonds betweenamino acids and is normally defined by the sequence of peptide-bondedamino acids and locations of disulfide bonds. The relative spatial arrangement of the linked amino acids is unspecified.Polypeptide chains are not free to take up any three-dimensionalstructure at random. Steric constraints and many weak interactionsstipulate that some arrangements will be more stable than others.Secondary structure refers to regular, recurring arrangements inspace of adjacent amino acid residues in a polypeptide chain. There area few common types of secondary structure, the most prominent beingthe a helix and the (3 conformation.
Tertiary structure refers to thespatial relationship among all amino acids in a polypeptide; it is thecomplete three-dimensional structure of the polypeptide. The boundary between secondary and tertiary structure is not always clear. Several different types of secondary structure are often found within thethree-dimensional structure of a large protein. Proteins with severalpolypeptide chains have one more level of structure: quaternarystructure, which refers to the spatial relationship of the polypeptides,or subunits, within the protein.Figure 7—2 Levels of structure in proteins.
Theprimary structure consists of a sequence of aminoacids linked together by covalent peptide bonds,and includes any disulfide bonds. The resultingpolypeptide can be coiled into an a helix, one formof secondary structure. The helix is a part of thetertiary structure of the folded polypeptide, which isitself one of the subunits that make up the quaternary structure of the multimeric protein, in thiscase hemoglobin.PrimarystructureSecondarystructureTertiarystructureQuaternarystructureAminoacidsa HelixPolypeptidechainAssembledsubunitsContinued advances in the understanding of protein structure,folding, and evolution have made it necessary to define two additionalstructural levels intermediate between secondary and tertiary structure.
A stable clustering of several elements of secondary structure issometimes referred to as supersecondary structure. The term isused to describe particularly stable arrangements that occur in many162Part II Structure and CatalysisFigure 7-3 The different structural domains inthe polypeptide troponin C, a calcium-binding protein associated with muscle. The separate calciumbinding domains, indicated in blue and purple, areconnected by a long a helix, shown in white.different proteins and sometimes many times in a single protein. Asomewhat higher level of structure is the domain.
This refers to acompact region, including perhaps 40 to 400 amino acids, that is adistinct structural unit within a larger polypeptide chain. A polypeptide that is folded into a dumbbell-like shape might be considered tohave two domains, one at either end. Many domains fold independently into thermodynamically stable structures. A large polypeptidechain can contain several domains that often are readily distinguishable within the overall structure (Fig. 7-3). In some cases the individual domains have separate functions.
As we will see, important patterns exist at each of these levels of structure that provide clues tounderstanding the overall structure of large proteins.A Protein's Conformation Is StabilizedLargely by Weak InteractionsThe native conformation of a protein is only marginally stable; thedifference in free energy between the folded and unfolded states intypical proteins under physiological conditions is in the range of only20 to 65 kJ/mol. A given polypeptide chain can theoretically assumecountless different conformations, and as a result the unfolded state ofa protein is characterized by a high degree of conformational entropy.This entropy, and the hydrogen-bonding interactions of many groupsin the polypeptide chain with solvent (water), tend to maintain theunfolded state. The chemical interactions that counteract these effectsand stabilize the native conformation include disulfide bonds and theweak (noncovalent) interactions described in Chapter 4: hydrogenbonds, and hydrophobic, ionic, and van der Waals interactions.
An appreciation of the role of these weak interactions is especially importantto understanding how polypeptide chains fold into specific secondary,tertiary, and quaternary structures.Every time a bond is formed between two atoms, some free energyis released in the form of heat or entropy. In other words, the formationof bonds is accompanied by a favorable (negative) change in free energy. The AG for covalent bond formation is generally in the range of-200 to -460 kJ/mol. For weak interactions, AG = - 4 to - 3 0 kJ/mol.Although covalent bonds are clearly much stronger, weak interactionspredominate as a stabilizing force in protein structure because of theirnumber.
In general, the protein conformation with the lowest free energy (i.e., the most stable) is the one with the maximum number ofweak interactions.The stability of a protein is not simply the sum of the free energiesof formation of the many weak interactions within it, however. Wehave already noted that the stability of proteins is marginal. Everyhydrogen-bonding group in a polypeptide chain was hydrogen bondedto water prior to folding. For every hydrogen bond formed in a protein,hydrogen bonds (of similar strength) between the same groups andwater were broken. The net stability contributed by a given weak interaction, or the difference in free energies of the folded and unfoldedstate, is close to zero.
We must therefore explain why the native conformation of a protein is favored. The contribution of weak interactions toprotein stability can be understood in terms of the properties of water(Chapter 4). Pure water contains a network of hydrogen-bonded watermolecules. No other molecule has the hydrogen-bonding potential ofwater, and other molecules present in an aqueous solution will disruptChapter 7 The Three-Dimensional Structure of Proteinsthe hydrogen bonding of water to some extent.
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