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Stein, J. Note that, as the disulfides are reduced, the b-mercaptoethanol is oxidized and forms dimers. When ribonuclease was treated with b -mercaptoethanol in 8 M urea, the product was a fully reduced, randomly coiled polypeptide chain devoid of enzymatic activity. When a protein is converted into a randomly coiled peptide without its normal activity, it is said to be denatured Figure 2. Anfinsen then made the critical observation that the denatured ribonu- clease, freed of urea and b -mercaptoethanol by dialysis Section 3.
He perceived the significance of this chance finding: the sulfhydryl groups of the denatured enzyme became oxidized by air, and the enzyme spontaneously refolded into a catalytically active form. Detailed studies then showed that nearly all the original enzymatic activity was regained if the sulfhydryl groups were oxidized under suitable condi- tions.
These experiments showed that the information needed to specify the catalytically active structure of ribonuclease is contained in its amino acid sequence.
Subsequent studies have established the generality of this central principle of biochemistry: sequence specifies conformation.
The dependence of confor- mation on sequence is especially significant because of the intimate connec- tion between conformation and function. A quite different result was obtained when reduced ribonuclease was reoxidized while it was still in 8 M urea and the preparation was then dia- lyzed to remove the urea. Why were the outcomes so different when reduced ribonuclease was reoxidized in the presence and absence of urea?
The reason is that the wrong disulfides formed pairs in urea. There are different ways of pairing eight cysteine molecules to form four disulfides; only one of these combinations is enzymatically active.
Anfinsen found that scrambled ribonuclease spontaneously converted into fully active, native ribonuclease when trace amounts of b -mercaptoethanol were added to an aqueous solution of the protein Figure 2.
The added b -mercaptoethanol catalyzed the rearrangement of disulfide pairings until the native structure was regained in about 10 hours. This process was driven by the decrease in free energy as the scrambled conformations were converted into the stable, native conformation of the enzyme.
The native disulfide pairings of ribonuclease thus contribute to the stabilization of the thermodynamically preferred structure. Similar refolding experiments have been performed on many other pro- teins. In many cases, the native structure can be generated under suitable conditions. For other proteins, however, refolding does not proceed efficiently. In these cases, the unfolded protein molecules usually become tangled up with one another to form aggregates.
Inside cells, proteins called chaperones block such undesirable interactions. Native ribonuclease can be re-formed from scrambled ribonuclease in the presence of a trace of b-mercaptoethanol. How does an unfolded polypeptide chain acquire the form of the native protein?
These fundamental questions in biochemistry can be approached by first asking a simpler one: What determines whether a par- ticular sequence in a protein forms an a helix, a b strand, or a turn? One source of insight is to examine the frequency of occurrence of particular amino acid residues in these secondary structures Table 2.
Residues such as alanine, glutamate, and leucine tend to be present in a helices, whereas valine and isoleucine tend to be present in b strands. Glycine, asparagine, and proline are more commonly observed in turns. Studies of proteins and synthetic peptides have revealed some reasons for these preferences. Branching at the b -carbon atom, as in valine, threo- nine, and isoleucine, tends to destabilize a helices because of steric clashes.
These residues are readily accommodated in b strands, where their side chains project out of the plane containing the main chain. Serine and aspar- agine tend to disrupt a helices because their side chains contain hydrogen- bond donors or acceptors in close proximity to the main chain, where they compete for main-chain NH and CO groups.
Can we predict the secondary structure of a protein by using this knowledge of the conformational preferences of amino acid residues? TABLE 2. Source: T. Creighton, Proteins: Structures and Molecular Properties, 2d ed.
Freeman and Company, , p. What stands in the way of more-accurate prediction? Note that the conformational preferences of amino acid residues are not tipped all the way to one structure Table 2. For example, glutamate, one of the strongest helix formers, prefers a helix to b strand by only a factor of three. The preference ratios of most other residues are smaller.
Indeed, some penta- and hexapeptide sequences have been found to adopt one structure in one protein and an entirely different structure in another Figure 2.
Kiganris Evolutionary Perspective Bioqkimica is evident in the structures and pathways of biochemistry, and is woven into the narrative of the textbook. In this edition, as in all previous editions, the authors thoroughly revised the text with an eye for clarity, rewriting and reorganizing discussions where advances in the field have given us a different perspective on biochemistry. Pathways and processes are presented in a physiological context so that the reader can see how biochemistry works in different parts of the body and under different environmental and hormonal conditions. Reverte Ediciones August Language: A straightforward and logical organization leads the reader through processes and helps navigate complex pathways and mechanisms. Janice Donnola Photo Editor: Problems and resources from the printed textbook are incorporated throughout the eBook, to ensure that students can easily review specific concepts. Your recently viewed items and featured recommendations.
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