I The Protein Foldmg Problem In theoly, all one needs to know in order to fold a protein into its biolo@ca& active shape is the sequence of its constituent amino acids. PVh,v has nobody been able to put theory into practice? by Frederic M. Richards n the late 1950s Christian E. Anfin- sen and his coIleagues at the Na- tional Institutes of Health made a remarkable discovery. They were explor- hlg a long-standing puzzle in biology: What causes newly made proteins- VtJhich resembk loosdy coiled smgs and are inactive-to wind into specifi- cally shaped balls able to perform cm- cia1 tasks in a living cell? In the process the team found the answer was simpler than anyone had ima,@ned. It seemed the amino acid sequence of a protein, a one-dimensional trait. was fully sufficient to speczy the mole- cule’s ulfimate three-dimensional shape and biological activity. (Proteins ar.e built from a set of just 20 amino acids, which are assembled into a chain ac- cording to directions embedded in the genes.) Outside factors, such as en- zymes that might catalyze folding, did not have to be invoked as mandatory participants. The discovery. which has since been commed n?my times-at least for rel- atively small proteins-suggested that the forces most responsible for proper folding in the ceil could, in theory, be derived From the basic principles of chemistry and physics. That is, if one knew the amino acid sequence of a protein, all that would have to be con- sidered would be the properties of the individual amino acids and their behav- ior in aqueous solution. (The interior of most CeUs is 70 to 90 percent water.) IR actuality, predicting the confor- mation of a protein on the basis of its amino acid sequence is far from sim- ple. More than 30 years after Adinsen made his breakthrough, hundreds of investigators are still at work on that challenge, which has come to be widely Professor of Molecular Biophysics and Hiochemistqr at Yale U1u\vrsiV. Richards joincd the Yale faculty in 1955. three years after earning his doctome from Harvard University. 54 SClENmlC AMERICAN January 1991 known as the protein folding problem. ‘The solution is of more than academ- ic interest. Many major hoped-for prod- ucts of the developing biotechnology industry are novel proteins. It is already possible to design genes to dircct the synthesis of such proteins. Yet failure to fold properly is a comon produc- tion concern. For a time, those of us working on the folding problem despaired of ever finding an answer. More recently, how- ever, advances in theory and experi- ment, combined with growing interest on the part of industry, have kindled new optimism. Most of the detailed information available so far comes from studies on small, water-soluble, globular proteins containing fewer th 300 or so amino acids. The relative importance of vari- ous rules of folding and assembly may be somewhat different for those pro- teins than for others-notably long fi- brous proteins and varieties residing in cellular membranes. Indeed. some large proteins have recently been shown to need foldhg help from other proteins known as chaperonins. The balance of the article will not consider such corn- plesities but will focus entirely on the unassisted folding reaction undergone by a great many proteins. It would be wonderful if research- ers had an atomic-level microscope that couid take a movie of individu- ai protein molecules folding up from their extended, unstable state to their final. or native, state, which is more stable. From a collection of qovies, all aspects of the reaction pathways could be seen directly. Unfortunately, no such instrument exists; investigators must Fall back on much less direct measure- ments and very car& reasoning. One can gather helpful clues to the rules of folding by examining the three- dimensional shuctures of unfolded andfully folded proteins and by analyz- illg [hc properties of individual amino acjds and small pcptidcs (lincar chains of amino acids). Fortunatcly, the archi- tecture of hundreds of native proteins has been determined by such imag- ing techniques as X-ray crystallography and, more recently, nuclear magnetic resonance (NMR). Both techniques have advanced dramatically in thc past dec- ade, as has thcoretical work attcrnpt- ing to predict folding rnathcmatically by computer. Isolatcd amino acids consist of a cen- tral carbon atom-called the alpha car- bon-bound to an amino group (NH.,), a carboxyl group (COOH) and a si& chain. The differences among amino acids, then, stem from differences in their side chains, namely, in shape, size and polarity. Shape and size affect the packing together of amino acids in the final molecule. Polarity (or lack of it) determines the nature and strength of interactions between amino acids in a protein and beween the protein and water. For instance, polar amino acids inter- act strongly with one another in what are called electrostatic interacaons. The molecules are considered polar if they carry a formal charge (owing to the loss or gain of one or more electrons) or if they are electrically neutral over- all but have localized regions where positive or negative charges predom- inate. {Positive charges are contribut- ed by protons in atomic nuclei. neg- ative charges by electrons surround- ing the nuclei.) Molecules are attracted when thcir oppositely charged regions arc closc; thcy arc rcpcllcd whcn like charged regions arc close. Nonpolar amino acids can also at- tract or repel one anothcr. albeit more wcakly. because of what are callcd van dcr Waals forces. Electrons and pro- tons vibratc constantly, and the vibra- tions result in attractions between sub- stances that are near one another. Thc attraction turns into repulsion whcn thc substances arc about to touch. In aqueous solution, polar amino acids tend to be hydrophilic; they at- tract water molcculcs, which are quite polar. In contrast, nonpolar amino ac- ids, which generally include hydrocar- bon side chains, tend to be hydropho- bic: they mix poorly with water and "prefer" to associate with one other. Alternatively, one can think of them as being squeezed out of the water as a consequence of the strong attraction between polar substances. The peptide