Proteins are the stuff of life. They are the eyes, arms and legs of living cells. Even DNA, the most iconic of all molecules in biology, is important first and foremost because it contains the genes that specify the makeup of proteins. And the cells in our body differ from one another—serving as neurons, white blood cells, smell sensors, and so on—largely because they activate different sets of genes and thus produce different mixtures of proteins.
Given these molecules’ importance, one would think biologists would have long figured out the basic picture of what they look like and how they work. Yet for decades scientists embraced a picture that was incomplete. They understood, quite properly, that proteins consist of amino acids linked together like beads on a string. But they were convinced that for a protein to function correctly, its amino acid chain first had to fold into a precise, rigid configuration. Now, however, it is becoming clear that a host of proteins carry out their biological tasks without ever completely folding; others fold only as needed. In fact, perhaps as many as one third of all human proteins are “intrinsically disordered, ” having at least some unfolded, or disordered, parts.
To be sure, biologists have known for a while that enzymes such as the polymerases that copy DNA or transcribe it into RNA are complicated nanomachines consisting of many moving parts, with hinges that allow different segments of a protein to pivot around one another. But those proteins are often pictured as combinations of rigid parts, like the sections of a folding chair. Intrinsically disordered proteins look more like partially cooked spaghetti constantly jiggling in a pot of boiling water.
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Fifteen years ago this assertion would have seemed downright heretical. Today scientists are realizing that such amorphous and flexible features probably helped life on earth get started and that their flexibility continues to play critical roles in cells, for instance, during cell division and gene activation. And this new understanding offers more than startling new insights into the basic biology of cells. Equally exciting, it hints at new ways for treating disease, including cancer.
The notion that a rigid, three-dimensional structure determines a protein’s function first emerged in 1894. Emil Fischer, a chemist at the University of Berlin, proposed that enzymes—the catalysts of biochemical reactions—interact with other molecules by binding to specific shapes on their outer surface; at the same time, enzymes would completely ignore any molecules whose surface features are only slightly different. In other words, an enzyme and its binding partner fit together like a key and a lock.
At the time Fischer formulated his model, the nature of proteins was unknown. Over the next 60 or so years biologists learned that proteins were chains of amino acids and concluded that they had to fold into a precise shape to work properly. In 1931 Chinese biochemist Hsien Wu lent strong support to that view, showing that protein denaturation, or loss of natural 3-D structure, led to a complete loss of function. Since then, starting with the 3-D structure of sperm whale myoglobin in 1958, researchers have determined the architecture of more than 50, 000 types of protein, usually by first coaxing their rigid structure into forming crystals and then scattering x-rays off those crystals.
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Not all was static in this structured, lock-and-key protein world, though. As far back as the early 1900s, scientists knew that many antibodies can bind to multiple targets, or antigens—an observation that did not fit neatly with the lock-and-key model. In the 1940s the great chemist Linus Pauling speculated that certain antibodies can fold up in any of several ways, with the folding of each configuration guided by the fit between antibody and antigen.
From about the 1940s on, various other observations indicated that not all proteins abided by the dogma that function follows from a rigid, 3-D structure. But those that did not were usually regarded as isolated, freak exceptions to the rule. One of us (Dunker) was among the first researchers to collect such examples and to note that perhaps the dogma itself needed revision. In 1953, for instance, scientists noticed that the milk protein casein is largely unstructured; this pliability probably facilitates its digestion by infant mammals. In the early 1970s a protein called fibrinogen was found to contain a region of significant size having no fixed structure; this region, along with similar but smaller ones discovered later, plays a key role in blood clotting. Later in the 1970s, the protein that forms the outer casing, or capsid, of the tobacco mosaic virus offered another striking example. When the capsid is empty, the protein has large, unstructured regions hanging loose inside the capsid’s cavity; that looseness enables newly minted RNA, made during viral reproduction in an infected cell, to pack inside. But as the RNA gets in, the protein binds to it and sets into a rigid shape.
Meanwhile experimenters who could not induce certain proteins to fold in their test tubes assumed they were doing something wrong: surely the amino acid chains would find a “correct” folded shape in the environment of the cell. For example, when researchers placed solutions containing isolated proteins into vials and scanned them with a nuclear magnetic resonance (NMR) spectrometer—a workhorse of protein studies—they would sometimes get blurry data, which they interpreted as indicating that the proteins had failed to fold.
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But those data had a richer story to tell. NMR spectroscopy involves the application of powerful radio-frequency pulses to induce the atomic nuclei of particular elements, such as hydrogen, to spin in sync. Slight frequency shifts in the nuclei’s response correlate tightly to the atoms’ positions inside amino acids and to the positions of those amino acids with respect to one another. Thus, from these frequency shifts investigators can often piece together the structure of a rigid protein. But if the amino acids move a lot—as would be the case in an unfolded protein—the frequency shifts become blurry.
In 1996 one of us (Kriwacki, then at the Scripps Research Institute) was performing NMR spectroscopy on a protein called p21, involved in controlling cell division, when he noticed something shocking. According to his NMR data, p21 was almost entirely disordered. The amino acids freely rotated about the chemical bonds that held them together, never staying in one conformation for more than a fraction of a second. And yet—and this was the shocking part—p21 was still able to perform its critical regulatory function. It was the first convincing demonstration that lack of structure does not make a protein useless.
NMR spectroscopy remains the primary technique to determine whether a protein is folded or disordered, and together with other technologies it has now confirmed that many proteins are intrinsically disordered. These molecules constantly morph under the action of Brownian motion and their own thermal jitters, and yet they are perfectly functional.
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This new, broader view is well illustrated by the protein p27, which is known to exist in most vertebrates. Like p21, p27 is one of the crucial proteins that regulate cell division so that cells do not multiply uncontrollably. NMR shows that p27 is highly flexible, with sections that rapidly fold and unfold into short-lived corkscrew- or sheet-shaped structures. Most cancer cells in humans have reduced amounts of p27, and the greater the loss, the poorer the prognosis for a patient’s survival.
The p27 molecule acts as a brake on cell division by binding to and inhibiting the activities of at least six different types of kinase enzymes. Kinases are the master regulators of DNA replication and cell division. They attach phosphate (PO
) to other proteins (“phosphorylate” them), a move that sets off a cascade of events. In carrying out its task, the stringlike, dynamic p27 molecule wraps around a kinase—which has a mostly rigid structure—and covers a significant portion of its surface, including its chemically reactive, or “active, ” sites. This blockage prevents phosphorylation and so arrests cell division. Thanks to its flexibility, then, p27 can mold itself around, and inhibit, different types of enzymes. Proteins with such an ability are described as promiscuous or moonlighting.
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The p27 protein, being almost completely unstructured, falls near the disordered end of a scale that ranges from complete disorder (totally unstructured) to complete order (totally rigidly folded). The kinases themselves fall near the opposite end of this scale. Many other proteins lie somewhere in between, having both structured and unstructured regions. Calcineurin, which is involved in immune responses (and is the target of antirejection drugs), is the reverse of a kinase: it removes phosphates from particular proteins that have been phosphorylated. It has a structured region that is the enzyme’s active site and operates in the classic lock-and-key-manner to remove phosphates from other proteins. But it also has an unstructured region that binds to and inactivates the enzyme’s own active site when phosphate removal is not needed. Thus, calcineurin is like two proteins in one: the structured region performs catalysis, and the unstructured region regulates this catalytic function.
The examples we have discussed so far are proteins that fold—either on themselves or
But those data had a richer story to tell. NMR spectroscopy involves the application of powerful radio-frequency pulses to induce the atomic nuclei of particular elements, such as hydrogen, to spin in sync. Slight frequency shifts in the nuclei’s response correlate tightly to the atoms’ positions inside amino acids and to the positions of those amino acids with respect to one another. Thus, from these frequency shifts investigators can often piece together the structure of a rigid protein. But if the amino acids move a lot—as would be the case in an unfolded protein—the frequency shifts become blurry.
In 1996 one of us (Kriwacki, then at the Scripps Research Institute) was performing NMR spectroscopy on a protein called p21, involved in controlling cell division, when he noticed something shocking. According to his NMR data, p21 was almost entirely disordered. The amino acids freely rotated about the chemical bonds that held them together, never staying in one conformation for more than a fraction of a second. And yet—and this was the shocking part—p21 was still able to perform its critical regulatory function. It was the first convincing demonstration that lack of structure does not make a protein useless.
NMR spectroscopy remains the primary technique to determine whether a protein is folded or disordered, and together with other technologies it has now confirmed that many proteins are intrinsically disordered. These molecules constantly morph under the action of Brownian motion and their own thermal jitters, and yet they are perfectly functional.
Synthetic Paper Separates Plasma From Whole Blood With Low Protein Loss
This new, broader view is well illustrated by the protein p27, which is known to exist in most vertebrates. Like p21, p27 is one of the crucial proteins that regulate cell division so that cells do not multiply uncontrollably. NMR shows that p27 is highly flexible, with sections that rapidly fold and unfold into short-lived corkscrew- or sheet-shaped structures. Most cancer cells in humans have reduced amounts of p27, and the greater the loss, the poorer the prognosis for a patient’s survival.
The p27 molecule acts as a brake on cell division by binding to and inhibiting the activities of at least six different types of kinase enzymes. Kinases are the master regulators of DNA replication and cell division. They attach phosphate (PO
) to other proteins (“phosphorylate” them), a move that sets off a cascade of events. In carrying out its task, the stringlike, dynamic p27 molecule wraps around a kinase—which has a mostly rigid structure—and covers a significant portion of its surface, including its chemically reactive, or “active, ” sites. This blockage prevents phosphorylation and so arrests cell division. Thanks to its flexibility, then, p27 can mold itself around, and inhibit, different types of enzymes. Proteins with such an ability are described as promiscuous or moonlighting.
Zoology Worksheets & Facts
The p27 protein, being almost completely unstructured, falls near the disordered end of a scale that ranges from complete disorder (totally unstructured) to complete order (totally rigidly folded). The kinases themselves fall near the opposite end of this scale. Many other proteins lie somewhere in between, having both structured and unstructured regions. Calcineurin, which is involved in immune responses (and is the target of antirejection drugs), is the reverse of a kinase: it removes phosphates from particular proteins that have been phosphorylated. It has a structured region that is the enzyme’s active site and operates in the classic lock-and-key-manner to remove phosphates from other proteins. But it also has an unstructured region that binds to and inactivates the enzyme’s own active site when phosphate removal is not needed. Thus, calcineurin is like two proteins in one: the structured region performs catalysis, and the unstructured region regulates this catalytic function.
The examples we have discussed so far are proteins that fold—either on themselves or
