The native-state ensemble of proteins provides cluesfor folding, misfolding and function

Nunilo Cremades1, Javier Sancho1 and Ernesto Freire2

1 Department of Biochemistry and BIFI, University of Zaragoza, Zaragoza, 50009, Spain2 Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA

494 Update TRENDS in Biochemical Sciences Vol.31 No.9

The predominant equilibrium in proteins is not betweennative and unfolded states, it is between the native andmultiple partially unfolded forms. Some of these par-tially unfolded forms can be energetically close to thenative state and, therefore, have the potential to becomeappreciably populated. This could have an importantrole in protein function or misfolding diseases. Therecent identification and characterization of the partiallyunfolded forms of apoflavodoxin furthers our under-standing of their formation.

The native-state ensembleThe study of the structural stability and folding of proteinshas attracted the attention of scientists for >70 years [1].Until recently, the methods available to evaluate proteinstability required the use of some form of denaturing agent(e.g. temperature, pH and chemical denaturants). Forsingle-domain proteins, equilibrium-denaturation methodsrevealed the presence of only two dominant conformations –the fully folded and the fully unfolded – which gave rise tothe classic ‘two-state’ hypothesis [2]. Within the ‘two-state’framework, partially folded structures have negligible prob-abilities and never become considerably populated.Although this is the case under denaturing conditions, itis not under native conditions. Itwas onlywith the advent ofthe technique of NMR-detected hydrogen–deuteriumexchange in the mid-1980s that the conformational equili-brium under native conditions could be investigated and anew view of the protein-folding equilibrium emerged (for areview, see Ref. [3]). The fundamental difference betweenNMR-detectedhydrogen–deuteriumexchangeandprevioustechniques is that it does not require the use of denaturingconditions and it enables determination of the stability ofindividual residues throughout theproteinstructure.Forallproteins thathavebeen studiedusing this technique todate,the view that has emerged is clear: under native conditions,proteins do not follow the two-state behavior. The predomi-nant equilibrium is not between the native and unfoldedstates but is, instead, between the native and several par-tially folded conformations. In the viewpoint pioneered byEnglander and co-workers [3,4], proteins undergo localizedunfolding reactions scattered throughout their entire struc-tures. These unfolding reactions give rise to states definedby the presence of one or several locally unfolded regions,termed ‘partially unfolded forms’ (PUFs). This collection ofstates defines the native-state ensemble, which can be

Corresponding author: Freire, E. (ef@jhu.edu).Available online 25 July 2006.

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computationally captured by approaches such as theCOREX algorithm [5]. This phenomenon is elegantlydemonstrated by Bollen et al. [6] for the apoflavodoxin ofAnabaena vinelandii.

ApoflavodoxinsApoflavodoxins are proteins of �170 residues that arearchetypical of the a/b class organized around a centralopen b sheet sandwiched between two helical layers; theyare ideally suited for folding and stability studies [6–8].Because flavodoxins are larger than most proteins cur-rently used in folding studies, they provide importantinsights into the behavior of proteins that are more repre-sentative of those found in nature. The experiments ofBollen et al. [6] culminate an important series of studiesthat indicate the existence of kinetic and equilibriumfolding intermediates in apoflavodoxins [7–9]. These inter-mediates seem to be a general feature of proteins thatexhibit flavodoxin-like folds as demonstrated experimen-tally [10] and predicted in computational studies [11,12].

Bollen et al. [6] demonstrate that the PUFs of apofla-vodoxin are off the folding pathway. Some of these PUFsare energetically close to the native state and, therefore,can become dominant under certain conditions. In protein-folding studies, changes in solution conditions such as lowpH, moderate concentrations of denaturants or changes inphysical conditions (e.g. temperature or pressure) are rou-tinely used to increase the population of PUFs. Moreimportantly, from a physiological stand-point, PUFs canbe populated by mutations that specifically lower thestability of the native state relative to specific PUFs, orby ligands or peptides that preferentially bind to PUFs,thus, increasing the prospects of misfolding diseases. Insome cases – for example, the HIV-1 proteins gp120 andNef – states with largely unstructured regions are presentin large populations and are functionally important [13].These proteins interact with more than one cellular part-ner in a concertedmanner. Usually, regions that define thebinding epitope for downstream partners are unstructuredand only become structured and binding competent whenthe preceding partner binds and allosterically induces thestructuring of the binding site for the next partner [13]. Inother cases, such as the alkaline transition of cytochrome c,even scarcely populated PUFs have a functional role [14].

Functional implicationsUnder native conditions, the Gibbs energy of theunfolded state of apoflavodoxin from A. vinelandii is

Figure 1. Numerical simulation of the Gibbs energy of stabilization that can be

contributed by a specific ligand, X, as a function of the ratio of free ligand conc-

entration to binding dissociation constant, [X]/Kd. Ligand concentrations 250-fold

higher than the Kd can generate stabilization energies of �3.5 kcal mol�1. Stabili-

zation energies >3.5 kcal mol�1 become increasingly difficult to achieve as the

required ligand concentration becomes extremely high. Inset: the population of a

PUF as a function of its intrinsic Gibbs energy relative to that of the native state.

Any PUF with a Gibbs energy >5 kcal mol�1 is <0.03% populated. Ligand binding

or mutations can lower the PUF energy and increase its population.

Update TRENDS in Biochemical Sciences Vol.31 No.9 495

�10.6 kcal mol�1 higher than that of the native state [6].The four PUFs identified by Bollen et al. [6] are clusteredbetween 4.9 and 8.7 kcal mol�1 from the native state.These values are within the range of 5 � 2 kcal mol�1

observed for PUFs in other proteins [15], suggesting simi-lar patterns in different proteins.

Under physiological conditions, the population ofPUFs is often negligible. From a functional or pathologicalpoint of view, an important question to ask is not how

Figure 2. A general schematic diagram of the Gibbs energy scale of protein

conformational states. The unfolded state is usually�10 kcal mol�1 higher than the

native state. The majority of states, including native-like partially folded states,

have much higher energies than the unfolded state and, therefore, have a negli-

gible chance of becoming populated under normal physiological conditions.

Intermediate states with Gibbs energies <5 kcal mol�1 have a realistic chance of

becoming considerably populated by mutations or ligands.

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large the population of a PUF, but the cost of transformingit into a considerably populated species. Any state thatis >3 kcal mol�1 higher than the native state is <1%populated; however, there is a big difference betweena statethat is 3–5 kcal mol�1 away fromthenative stateanda statethat is 15 or more kcal mol�1 away. The difference residesin their potential for becoming populated by existing con-ditions in the organism. For example, how much energy aspecific ligand or a single-point mutation can contributetowards the stabilization of a PUF? The contribution of aspecific ligand can be easily calculated given its concentra-tion and binding affinity for the PUF (Figure 1). It is clearthat contributions up to 3 kcal mol�1 can be achieved,whereas higher contributions become progressively moredifficult to attain because the ligand concentration requiredbecomes prohibitive for the organism as it increases. A PUFthat is 5 kcal mol�1 away from the native state is only 0.03%populated at 37 8C. To become 1% or 5% populated, astabilizing DG of 2.2 or 3.2 kcal mol�1 is required. Thisenergy is within the range that can be provided by a ligand(Figure 1, inset) or by mutations.

In principle, proteins have astronomical numbers of pos-sible conformational states; however, only few have thepotential of being populated under normal physiologicalconditions (Figure 2). As for apoflavodoxin, the unfoldedstate ofmost proteins is usually�10 kcal mol�1 higher thanthe native state. The low-energy PUFS (<5 kcal mol�1) arethe ones that have a realistic chance of being populatedby conditions found in the organism. Nevertheless, evenhigh-energy PUFS might have functional roles, as shownexperimentally for cytochrome c [14].

Concluding remarksThe results of Bollen et al. [6] provide fundamental informa-tion about the folding pathway of apoflavodoxin; inaddition, this type of study might also prove crucial for abetter understanding of protein-function and protein-conformational diseases. The tools are available for proteinscientists to experimentally examine the functional andpathological roles of PUFs that can becomepopulatedundernativeconditionsand the specificmechanismsbywhich theyare stabilized. These advances should be invaluable inthe design of novel strategies to combat protein-foldingdiseases.

AcknowledgementsSupported by grants from the National Institutes of Health GM 57144and GM56550 and the National Science Foundation MCB0131241 (E.F.)and BFU2004–01411 (J.S.).

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6 Bollen, Y.J. et al. (2006) The folding energy landscape of apoflavodoxinis rugged: hydrogen exchange reveals nonproductive misfoldedintermediates. Proc. Natl. Acad. Sci. U. S. A. 103, 4095–4100

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