A series of articles for general audiences Unraveling the Mystery of Protein Folding by W. A. (Bill) Thomasson This series of essays was that allow our muscles to con- developed as part of FASEB’s tract, and the collagen that makes efforts to educate the general up our tendons and ligaments public, and the legislators whom (and even much of our bones)all it elects, about the benefits of are proteins. fundamental biomedical To make proteins, machines research—particularly how known as ribosomes string to- investment in such research gether amino acids into long, leads to scientific progress, linear chains. Like shoelaces, improved health, and economic these chains loop about each other well-being. in a variety of ways (i.e., they fold). But, as with a shoelace, Alzheimers disease. Cystic fibro- only one of these many ways sis. Mad Cow disease. An inher- allows the protein to function ited form of emphysema. Even properly. Yet lack of function is many cancers. Recent discoveries not always the worst scenario. show that all these apparently For just as a hopelessly knotted unrelated diseases result from shoelace could be worse than one protein folding gone wrong. As that wont stay tied, too much of a though that werent enough, many misfolded protein could be worse of the unexpected difficulties than too little of a normally folded biotechnology companies encoun- one. This is because a misfolded ter when trying to produce human protein can actually poison the proteins in bacteria also result cells around it. from something amiss when Early Studies proteins fold. What exactly is this phenom- The importance of protein folding enon? We all learned that pro- has been recognized for many teins are fundamental compo- years. Almost a half-century ago, nents of all living cells: our own, Linus Pauling discovered two quite the bacteria that infect us, the plants and animals we eat. The W. A. (Bill) Thomasson, Ph.D., is a science and medical writer based in Oak Park, IL. hemoglobin that carries oxygen to Jonathan A. King served as science writer. our tissues, the insulin that sig- This series is available on FASEBs Public nals our bodies to store excess Policy Home Page at sugar, the antibodies that fight http://www.faseb.org/opa/ or as reprints from FASEBs Office of Public Affairs, 9650 infection, the actin and myosin Rockville Pike, Bethesda, MD 20814. simple, regular arrangements of egg cools, the proteins dont re- amino acidsthe α-helix and the turn to their original shapes. β-sheet (see the box, Fundamental Instead, they form a solid, in- Patterns of Protein Structure) soluble (but tasty) mass. This is that are found in almost every misfolding. Similarly, biochemists protein. And in the early 1960s, have always cursed the tendency Christian Anfinsen showed that of some proteins to form the in- the proteins actually tie them- soluble lumps in the bottom of selves: If proteins become un- their test tubes. We now know folded, they fold back into proper that these, too, were proteins shape of their own accord; no folded into the wrong shapes. shaper or folder is needed. Until recently, biochemists Of course, neither Pauling nor lacked the tools to study these Anfinsen nor the committees that insoluble lumps. Nor did they awarded them their respective expect such masses would be Nobel prizes knew at the time particularly interesting. The that these discoveries would be so prevailing view at the time was important for understanding that the lumps were just hope- Alzheimers disease or cystic lessly tangled and completely fibrosis. And when Pauling, at amorphous masses of protein least, was doing his breakthrough fibers (aggregation). Researchers studies, he could hardly have eventually discovered that these imagined the enormity of todays aggregates of incorrect folding biotechnology industry. What could be highly structured, but scientists did know is that any before this crucial insight and process that was so fundamental before proper investigative tools to life as protein folding would were developed, biochemists have to be of the utmost practical simply threw their fouled test importance. tubes away. But research did not stop with Gunking Up’ Tissues Pauling and Anfinsen. Indeed, we now know that Anfinsens conclu- As far back as the start of this sions needed expansion: Some- century, physicians have been times a protein will fold into a noticing that certain diseases are wrong shape. And some proteins, characterized by extensive pro- aptly named chaperones, keep tein deposits in certain tissues. their target proteins from getting Most of these diseases are rare, off the right folding path (see the but Alzheimers is not. It was box, Molecular Chaperones). Alois Alzheimer himself who These two small but important noted the presence of neurofibril- additions to Anfinsens theory lary tangles and neuritic plaque hold the keys to protein folding in certain regions of his patients diseases. brain. Tangles are more or less Weve known since antiquity common in diseases that feature (but didnt know we knew) that extensive nerve cell death; plaque, protein folding can go wrong. however, is specific to Alzheimers. When we boil an egg, the proteins The major question, which has in the white unfold. But when the only recently been answered, is Fundamental Patterns of Protein Structure More than a half century ago, evidence began to accumulate that a major part of most proteins folded structure consists of two regular, highly periodic arrangements, designated α and β. In 1951 researchers worked out the precise nature of these arrangements. The key to both structures is the hydrogen bond. A hydrogen atom is nothing more than a proton with a surrounding electron cloud. When one of these atoms is chemically bonded to an electron-withdrawing atom such as nitrogen or oxygen, much of the electron cloud moves toward the nitrogen or oxygen. The proton is thus left almost bare, with its positive charge largely unshielded. If it comes close to another atom with a bit of ex- tra negative charge (typi- cally, an oxygen or nitrogen atom), the par- tial positive and negative charges will attract each other. It is this attraction that produces the hydrogen bond and stabilizes the α and β structures. The a struc- ture, nw called α-helix, is a right-hand spiral stabilized by hydrogen bonds (A) A model alpha-helix shows the hydrogen bonds (dotted lines) between oxygen and hydrogen atoms of the between each fourth amino acid up the chain. (B) beta-sheets are also held together by hydrogen bonds. The transparent amino acids ni- arrows show the direction of individual beta-strands. Chains running in the same direction (left pair) are trogen atom called parallel beta sheet; strands running in opposite directions (right pair) are said to be anti-parallel and the oxygen beta-sheet. The atom coloring is as follows: carbon = green, oxygen = red, nitrogen = blue, and white = atom of the hydrogen. (Courtesy: Stanley Krystek, Bristol-Myers Squibb, Pharmaceutical Research Institute) fourth one up the chain. This means that there are 3.6 amino acids for each turn of the helix. The main part of the amino acid (the side chain, designated R in the figure) sticks out from this spiral backbone like the bristles on a bottle brush. The β structure is now called β-sheet. It is essentially flat, with the side chains sticking out on alternate sides. β-sheet is also stabilized by hydrogen bonds between nitrogen and oxygen atoms. In this case, however, the hydrogen-bonded atoms belong to different amino acid chains running alongside each other. The sheets are parallel if all the chains run in the same direction and are antiparallel if alternate chains run in opposite directions. Antiparallel sheets are often, but not always, formed by a single chain looping back upon itself. When a single chain loops back on itself to form an antiparallel β-sheet, the one to three amino acids linking the two strands are known as a β-turn. Today, scientists recognize the β-turn as one of the funda- mental elements of protein structure. All other local arrangements of amino acids are described as ran- dom coil, although they are random only in the sense of not being periodic. whether plaque causes millions of potential folded states Alzheimers or, like tangles, is a to choose from, consistently found consequence of it. the correct oneand did so within Further investigation showed seconds to minutes. that neuritic plaque (unrelated to Could there be specific, critical the plaque that clogs atheroscle- intermediates (partially folded rotic blood vessels and causes chains) in the folding process? heart attacks) is composed almost This turned out to be a difficult entirely of a single protein. De- question to answer. Partially posits of large amounts of a folded chains dont stay that way single, insoluble protein around very long; they become fully the degenerating nerve cells of folded chains in a fraction of a Alzheimers disease eventually second. Nevertheless, by the provided a key to understanding early 1980s researchers had not the disorder. only found clear evidence for the It was development of the bio- existence of partially folded pro- technology industry that unex- teins, but also realized the key pectedly spurred interest in in- role these played in the folding soluble protein gunk. This indus- process. try can produce proteins (often One study involved the diffi- otherwise difficult-to-obtain hu- culty in getting bovine growth man proteins) quickly and eco- hormone to fold properly. Al- nomically in bacteria. To their though the unfolded proteins were surprise, however, scientists who not sticky, and the fully folded worked for biotech companies proteins were not sticky, the often found two things: protein partially folded molecules stuck to that was supposed to be soluble each othera first clue as to the instead precipitated as insoluble origins of misfolded lumps (at inclusion bodies within the bacte- least for purified proteins in test ria and proteins that were sup- tubes).