Converging concepts of protein folding in vitroand in vivoF Ulrich Hartl & Manajit Hayer-HartlMost proteins must fold into precise three-dimensional conformations to fulfill their biological functions. Here we review recentconcepts emerging from studies of protein folding in vitro and in vivo, with a focus on how proteins navigate the complex foldingenergy landscape inside cells with the aid of molecular chaperones. Understanding these reactions is also of considerable medicalrelevance, as the aggregation of misfolding proteins that escape the cellular quality-control machinery underlies a range ofdebilitating diseases, including many age-onset neurodegenerative disorders.Numerous proteins have been shown to fold spontaneously in vitro,confirming Anfinsen’s pioneering insight that the linear sequence ofthe polypeptide chain contains all the necessary information to specifya protein’s three-dimensional structure1. Although protein folding hasbeen studied intensely for almost 50 years, how the final fold (and thefolding process) is determined by the amino acid sequence remainsone of the most important problems in biology. Moreover, in themore recent past it has become clear that, in the cell, a large fraction ofnewly synthesized proteins require assistance by molecular chaperonesto reach their folded states efficiently and on a biologically relevanttimescale2. Clearly, proteins in the test tube and in the cell are subjectto the same laws of physics, so what is special about folding undercellular conditions, and why are chaperones necessary? The increasingavailability of highly sensitive biophysical techniques to study foldingin vitro and in cellular systems is now providing new insights intothese issues (see the Review by Bartlett and Radford3in this issue).These studies also shed light on the process of aggregation, apotentially dangerous off-pathway reaction that can cause diseaseand must be prevented by molecular chaperones.Folding and aggregationFolding intermediates are the rule for larger proteins of 4100 aminoacids (B90% of all proteins in a cell), which have a greater tendencyto rapidly collapse in aqueous solution into compact non-nativeconformations4. As shown recently by a combination of rapid mixingtechniques and sensitive spectroscopic measurements, even smallproteins that fold on a subsecond timescale may pass throughstructural intermediates en route to the native state3,4. Such inter-mediates either represent on-pathway ‘stepping stones’ toward thenative state or kinetically stable, misfolded conformations that mayrequire substantial reorganization before the native state can bereached. The formation of metastable, non-native interactions duringfolding is interpreted as a consequence of the ruggedness of thefunnel-shaped folding energy landscape5,6(Fig. 1), irrespective ofwhether proteins are thought to fold through multiple downhill routesor through preferred pathways defined by the sequential assembly ofelementary folding units, so-called ‘foldons’7,8. Examples of suchminimal nucleation motifs are the two-stranded-helix motifs foundin a/b domain proteins. Multistate folding behavior with populatedintermediates would be observed when multiple foldons are separatedand do not act cooperatively8or when foldons misassemble, resultingin a kinetic block of folding. The propensity to misfold increases withtopologically complex fold types that are stabilized by long-rangeinteractions (for example, a/b domain architectures) or when proteinscontain multiple domains that are separate in the native state but mayinteract during folding9,10.Partially folded or misfolded states often tend to aggregate, parti-cularly when they represent major kinetic traps in the folding pathway.This is due to the fact that these forms typically expose hydrophobicamino acid residues and regions of unstructured polypeptide back-bone, features that are mostly buried in the native state. Likeintramolecular folding, aggregation—the association of two or morenon-native protein molecules—is largely driven by hydrophobic forcesand primarily results in the formation of amorphous structures(Fig. 1). Alternatively, aggregation can lead to the formation of highlyordered, fibrillar aggregates called amyloid, in which b-strands runperpendicular to the long fibril axis (cross-b structure) (Fig. 1).Although apparently restricted to a subset of proteins under physio-logical conditions, these thermodynamically highly stable structuresare accessible to many proteins under denaturing conditions, largelyindependent of sequence, suggesting that their formation is aninherent property of the polypeptide chain11. The formation ofamyloid fibrils is usually toxic to cells and may give rise to some ofthe most debilitating neurodegenerative diseases.Folding in the cell—the molecular chaperone conceptNew, fluorescence-based techniques now allow protein folding andaggregation to be observed in vivo in real time12. These and otherstudies indicate that the tendency of partially folded proteins toPublished online 3 June 2009; doi:10.1038/nsmb.1591Max Planck Institute of Biochemistry, Department of Cellular Biochemistry,Martinsried, Germany. Correspondence should be addressed to F.U.H.([email protected]) or M.H.-H. ([email protected]).574 VOLUME 16 NUMBER 6 JUNE 2009 NATURE STRUCTURAL & MOLECULAR BIOLOGYREVIEWPROTEIN FOLDING © 2009 Nature America, Inc. All rights reserved.aggregate is greatly enhanced in the highly crowded environment ofthe cell, largely explaining the requirement of molecular chaperones13.Whereas folding experiments in vitro are typically performed in dilutesolution to minimize aggregation, in the cell, folding occurs in thepresence of 300–400 g l–1of protein and other macromolecules. Theresulting excluded volume effects substantially enhance the affinitiesbetween interacting protein molecules, including folding intermedi-ates. The translation process can potentially further increase the risk ofmisfolding and aggregation, because incomplete polypeptide chainscannot fold into stable native conformations. Additionally, the exitchannel of the large ribosomal subunit, which is B100 A˚long but, atmost, 20 A˚wide14, largely precludes folding beyond the formation ofa-helical elements15,16and thus prevents the C-terminal 40–60 resi-dues of the chain from participating in long-range interactions (seethe Review by Bukau and colleagues17in this issue). As a consequence,productive