Measuring the stability of partly folded proteins using TMAO

151

242

Native molten globules are the most folded kind of intrinsically disordered proteins. Little is known about the mechanism by which native molten globules bind to their cognate ligands to form fully folded complexes. The nuclear coactivator binding domain (NCBD) of CREB binding protein is particularly interesting in this respect as structural studies of its complexes have shown that NCBD folds into two remarkably different states depending on the ligand being ACTR or IRF-3. The ligand-free state of NCBD was characterized in order to understand the mechanism of folding upon ligand binding. Biophysical studies show that despite the molten globule nature of the domain, it contains a small cooperatively folded core. By NMR spectroscopy, we have demonstrated that the folded core of NCBD has a well ordered conformer with specific side chain packing. This conformer resembles the structure of the NCBD in complex with the protein ligand, ACTR, suggesting that ACTR binds to prefolded NCBD molecules from the ensemble of interconverting structures.CREB binding protein | folding upon binding | NMR spectroscopy T he protein structure-function paradigm has dominated structural and molecular biology over the past five decades. This paradigm has been challenged by the discovery of functional but intrinsically disordered proteins (IDPs) (1). IDPs are abundant in higher organisms and are necessary for many crucial biological functions such as cell cycle regulation, signal transduction, and regulation of transcription (2, 3). Structural studies of IDPs have shown that despite the high degree of disorder, these proteins are far from randomly structured and form transient, yet specific, secondary and tertiary structural elements (4, 5). The success of the structure-function paradigm in explaining the function of folded proteins suggests that the functions of the IDPs may also be understood by studying the transient structure of the disordered state.The molten globule was originally discovered as a partially folded state under mildly denaturing conditions and was shown to accumulate as an intermediate during protein folding reactions (6, 7). With the emergence of the IDPs, proteins were discovered where the molten globule is the functionally active state. Native molten globules are the most compact conformational state considered as IDPs (4). Molten globules have native-like secondary structure, but are believed to lack the well-defined tertiary interactions found in folded proteins. Most high resolution NMR studies of the molten globule state have focused on the secondary structure of the backbone as this is more readily accessible from secondary chemical shifts (8). The degree of side chain packing, however, is less well understood. Molten globules are believed to have dynamic hydrophobic cores due to the lack of signal in the near-UV CD spectrum and the broadening of NMR signals of aromatic groups (6, 7). Recent studies have shown that the side chains in the α-lactalbumin molten globule exchange between several well defined confor...

Section: Resultsmentioning

confidence: 99%

Conformational selection in the molten globule state of the nuclear coactivator binding domain of CBP

Kjærgaard

Teilum

Poulsen

2010

151

242

“…The proteins used in this study were drawn from those reported in studies by Myers et al and Hong et al, along with some we added (7,(29)(30)(31)(32)(33)(34)(35). The m values depend on salt concentration and pH, so only those proteins are included that are monomeric, denature reversibly in the presence of urea at neutral pH and moderate salt, exhibit two-state behavior, and contain no cofactor or metal ion.…”

Section: Discussionmentioning

confidence: 99%

Anatomy of energetic changes accompanying urea-induced protein denaturation

Auton

Holthauzen

Bolen

2007

260

401

Because of its protein-denaturing ability, urea has played a pivotal role in the experimental and conceptual understanding of protein folding and unfolding. The measure of urea's ability to force a protein to unfold is given by the m value, an experimental quantity giving the free energy change for unfolding per molar urea. With the aid of Tanford's transfer model [Tanford C (1964) J Am Chem Soc 86:2050 -2059], we use newly obtained group transfer free energies (GTFEs) of protein side-chain and backbone units from water to 1 M urea to account for the m value of urea, and the method reveals the anatomy of protein denaturation in terms of residue-level free energy contributions of groups newly exposed on denaturation. The GTFEs were obtained by accounting for solubility and activity coefficient ratios accompanying the transfer of glycine from water to 1 M urea. Contrary to the opinions of some researchers, the GTFEs show that urea does not denature proteins through favorable interactions with nonpolar side chains; what drives urea-induced protein unfolding is the large favorable interaction of urea with the peptide backbone. Although the m value is said to be proportional to surface area newly exposed on denaturation, only Ϸ25% of the area favorably contributes to unfolding (because of newly exposed backbone units), with Ϸ75% modestly opposing urea-induced denaturation (originating from side-chain exposure). Use of the transfer model and newly determined GTFEs achieves the long-sought goal of predicting urea-dependent cooperative protein unfolding energetics at the level of individual amino acid residues. m value ͉ transfer free energy ͉ transfer model ͉ activity coefficient ͉ self-avoiding random coil U nderstanding the energetics of protein-solute interactions is one of the most elusive goals of protein science, with urea-induced denaturation serving as a long-standing reminder of the inability to experimentally account for such fundamental interactions in a detailed manner. More than 40 years ago, Tanford set out to identify the sites and free energies of urea interaction with protein groups in enough detail to account for the energetics of urea-induced denaturation (1-3). In principle, the transfer model he developed provides a means of dissecting which groups (side chain and backbone) are involved in denaturation and how much they contribute to the free energy of denaturation by urea. For the transfer model (see Scheme 1) to be successful, two requirements must be met: (i) accurate transfer free energy changes for side-chain and backbone groups must be known, and (ii) the free energy of transfer of a native or denatured state of a protein from water to the urea solution must be equal to the sum of the transfer free energy contributions of its solvent-exposed parts. Of these two requirements, the ability to obtain accurate transfer free energies of side chains and backbone groups has been a particularly difficult impediment to quantifying the energetics of urea-induced denaturation. Here, we present results fo...

“…Current experimental evidence points to hydrogen bonding. Specifically, at least four natively unfolded proteins can be forced to fold upon addition of organic osmolytes (87,120,121). In such cases, the total free energy of osmolyteinduced folding can be dissected into individual group transfer-free energies (86), whereupon it becomes evident that the peptide backbone is the dominant contributor.…”

Section: Part 7 Protein Folding: Analog Mechanism Vs Digital Mechanismmentioning

confidence: 99%

A backbone-based theory of protein folding

Rose

Fleming

Banavar

et al. 2006

448

412

Under physiological conditions, a protein undergoes a spontaneous disorder º order transition called "folding." The protein polymer is highly flexible when unfolded but adopts its unique native, three-dimensional structure when folded. Current experimental knowledge comes primarily from thermodynamic measurements in solution or the structures of individual molecules, elucidated by either x-ray crystallography or NMR spectroscopy. From the former, we know the enthalpy, entropy, and free energy differences between the folded and unfolded forms of hundreds of proteins under a variety of solvent͞cosolvent conditions. From the latter, we know the structures of Ϸ35,000 proteins, which are built on scaffolds of hydrogen-bonded structural elements, ␣-helix and ␤-sheet. Anfinsen showed that the amino acid sequence alone is sufficient to determine a protein's structure, but the molecular mechanism responsible for self-assembly remains an open question, probably the most fundamental open question in biochemistry. This perspective is a hybrid: partly review, partly proposal. First, we summarize key ideas regarding protein folding developed over the past half-century and culminating in the current mindset. In this view, the energetics of side-chain interactions dominate the folding process, driving the chain to self-organize under folding conditions. Next, having taken stock, we propose an alternative model that inverts the prevailing side-chain͞backbone paradigm. Here, the energetics of backbone hydrogen bonds dominate the folding process, with preorganization in the unfolded state. Then, under folding conditions, the resultant fold is selected from a limited repertoire of structural possibilities, each corresponding to a distinct hydrogen-bonded arrangement of ␣-helices and͞or strands of ␤-sheet.