Probing protein folding and stability using disulfide bonds

Disulfide bonds are required to stabilize the folded conformations of many proteins. The rates and equilibria of processes involved in disulfide bond formation and breakage can be manipulated experimentally and can be used to obtain important information about protein folding and stability. A number of experimental procedures for studying these processes, and approaches to interpreting the resulting data, are described here.     

Folding,stability, and secondary structure of a new dimeric cysteine proteinase inhibitor

Clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, is a 34-kDa homodimer lacking disulphide bonds, reported to have unusual stability properties. Sequence similarity is limited solely to certain proteins from mushrooms. Infrared spectroscopy shows that clitocypin is a high beta-structure protein which was lost at high temperatures. The far UV circular dichroism spectrum is not that of classical beta-structure, but similar to those of a group of small beta-strand proteins, with a peak at 189nm and a trough at 202nm. An aromatic peak at 232nm and infrared bands at 1633 and 1515cm(-1) associated with the peptide backbone and the tyrosine microenvironment, respectively, were used to characterize the thermal unfolding. The reversible transition has a midpoint at 67 degrees C, with DeltaG=34kJ/mol and DeltaH=300kJ/mol, and is, unusually, independent of protein concentration. The kinetics of thermal unfolding and refolding are slow, with activation energies of 167 and 44kJ/mol, respectively. A model for folding and assembly is discussed.     

Effect of surface electrostatic interactions on the stability and folding of formate dehydrogenase from Candida methylica

NAD⁺-dependent formate dehydrogenase (FDH-EC 1.2.1.2) is an important enzyme to regenerate valuable NADH required by NAD⁺-dependent oxidoreductases in enzyme catalysis. The limitation in the thermostability of FDH enzyme is a crucial problem for development of biotechnological and industrial processes, despite of its advantages. In this study, to investigate the contribution of surface electrostatic interaction to the thermostability of FDH from Candida methylica (cmFDH) N187E, H13E, Q105R, N300E, N147R N300E/N147R, N187E/Q105R, N187E/N147R,Y160R, Y302R, Y160E and Y302E mutants were designed using a homology model of cmFDH based on Candida boidinii (cb) by considering electrostatic interactions on the protein surface. The effects of site-specific engineering on the stability of this molecule was analyzed according to minimal model of folding and assembly reaction and deduced equilibrium properties of the native system with respect to its thermal and denaturant sensitivities. It was observed that mutations did not change the unfolding pattern of native cmFDH and increased numbers of electrostatic interactions can cause either stabilizing or destabilizing effect on the thermostability of this protein. The thermodynamic and kinetic results suggested that except relatively improved mutants, three out of the nine single mutations increased the melting temperature of cmFDH enzyme.     

Understanding the determinants of stability and folding of small globular proteins from their energetics

The results of minimal model calculations indicate that the stability and the kinetic accessibility of the native state of small globular proteins are controlled by few "hot" sites. By means of molecular dynamics simulations around the native conformation, which describe the protein and the surrounding solvent at the all-atom level, an accurate and compact energetic map of the native state of the protein is generated. This map is further simplified by means of an eigenvalue decomposition. The components of the eigenvector associated with the lowest eigenvalue indicate which hot sites are likely to be responsible for the stability and for the rapid folding of the protein. The comparison of the results of the model with the findings of mutagenesis experiments performed for four small proteins show that the eigenvalue decomposition method is able to identify between 60% and 80% of these (hot) sites.     

Conformational stability of dimeric and monomeric forms of the C-terminal domain of human immunodeficiency virus-1 capsid protein

The unfolding equilibrium of the C-terminal domain of human immunodeficiency virus-1 (HIV-1) capsid protein has been analyzed by circular dichroism and fluorescence spectroscopy. The results for the dimeric, natural domain are consistent with a three-state model (N(2)<-->2I<-->2U). The dimer (N(2)) dissociates and partially unfolds in a coupled cooperative process, into a monomeric intermediate (I) of very low conformational stability. This intermediate, which is the only significantly populated form at low (1 microM) protein concentrations, fully preserves the secondary structure but has lost part of the tertiary (intramonomer) interactions found in the dimer. In a second transition, the intermediate cooperatively unfolds into denatured monomer (U). The latter process is the equivalent of a two-state unfolding transition observed for a monomeric domain in which Trp184 at the dimer interface had been truncated to Ala. A highly conserved, disulfide-bonded cysteine, but not the disulfide bond itself, and three conserved residues within the major homology region of the retroviral capsid are important for the conformational stability of the monomer. All these residues are involved also in the association process, despite being located far away from the dimerization interface. It is proposed that dimerization of the C-terminal domain of the HIV-1 capsid protein involves induced-fit recognition, and the conformational reorganization also improves substantially the low intrinsic stability of each monomeric half.     

Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

Members of the phosducin gene family were initially proposed to act as down-regulators of G protein signaling by binding G protein βγ dimers (Gβγ) and inhibiting their ability to interact with G protein subunits (G) and effectors. However, recent findings have over-turned this hypothesis by showing that most members of the phosducin family act as co-chaperones with the cytosolic chaperonin complex (CCT) to assist in the folding of a variety of proteins from their nascent polypeptides. In fact rather than inhibiting G protein pathways, phosducin-like protein 1 (PhLP1) has been shown to be essential for G protein signaling by catalyzing the folding and assembly of the Gβγ dimer. PhLP2 and PhLP3 have no role in G protein signaling, but they appear to assist in the folding of proteins essential in regulating cell cycle progression as well as actin and tubulin. Phosducin itself is the only family member that does not participate with CCT in protein folding, but it is believed to have a specific role in visual signal transduction to chaperone Gβγ subunits as they translocate to and from the outer and inner segments of photoreceptor cells during light-adaptation.     

Strategies for folding of affinity tagged proteins using GroEL and osmolytes

Obtaining a proper fold of affinity tagged chimera proteins can be difficult. Frequently, the protein of interest aggregates after the chimeric affinity tag is cleaved off, even when the entire chimeric construct is initially soluble. If the attached protein is incorrectly folded, chaperone proteins such as GroEL bind to the misfolded construct and complicate both folding and affinity purification. Since chaperonin/osmolyte mixtures facilitate correct folding from the chaperonin, we explored the possibility that we could use this intrinsic binding reaction to advantage to refold two difficult-to-fold chimeric constructs. In one instance, we were able to recover activity from a properly folded construct after the construct was released from the chaperonin in the presence of osmolytes. As an added advantage, we have also found that this method involving chaperonins can enable researchers to decide (1) if further stabilization of the folded product is required and (2) if the protein construct in question will ever be competent to fold with osmolytes.     

Translocons, thermodynamics, and the folding of membrane proteins

Recent three-dimensional structures of helical membrane proteins present new challenges for the prediction of structure from amino acid sequence. Membrane proteins reside stably in a thermodynamic free energy minimum after release into the membrane's bilayer fabric from the translocon complex. This means that structure prediction is primarily a problem of physical chemistry. But the folding processes within the translocon must also be considered. A distilled overview of the physical principles of membrane protein stability is presented, and extended to encompass translocon-assisted folding.     

Measuring the stability of partly folded proteins using TMAO

Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that DeltaG degrees for unfolding depends linearly on TMAO concentration, and that the sensitivity of DeltaG degrees to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of DeltaG degrees on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.     

Unification of the folding mechanisms of non-two-state and two-state proteins

We have collected the kinetic folding data for non-two-state and two-state globular proteins reported in the literature, and investigated the relationships between the folding kinetics and the native three-dimensional structure of these proteins. The rate constants of formation of both the intermediate and the native state of non-two-state folders were found to be significantly correlated with protein chain length and native backbone topology, which is represented by the absolute contact order and sequence-distant native pairs. The folding rate of two-state folders, which is known to be correlated with the native backbone topology, apparently does not correlate significantly with protein chain length. On the basis of a comparison of the folding rates of the non-two-state and two-state folders, it was found that they are similarly dependent on the parameters that reflect the native backbone topology. This suggests that the mechanisms behind non-two-state and two-state folding are essentially identical. The present results lead us to propose a unified mechanism of protein folding, in which folding occurs in a hierarchical manner, reflecting the hierarchy of the native three-dimensional structure, as embodied in the case of non-two-state folding with an accumulation of the intermediate. Apparently, two-state folding is merely a simplified version of hierarchical folding caused either by an alteration in the rate-limiting step of folding or by destabilization of the intermediate.     

Proteomic,functional and motif‐based analysis of C‐terminal Src kinase‐interacting proteins

C‐terminal Src kinase (Csk) that functions as an essential negative regulator of Src family tyrosine kinases (SFKs) interacts with tyrosine‐phosphorylated molecules through its Src homology 2 (SH2) domain, allowing it targeting to the sites of SFKs and concomitantly enhancing its kinase activity. Identification of additional Csk‐interacting proteins is expected to reveal potential signaling targets and previously undescribed functions of Csk. In this study, using a direct proteomic approach, we identified 151 novel potential Csk‐binding partners, which are associated with a wide range of biological functions. Bioinformatics analysis showed that the majority of identified proteins contain one or several Csk‐SH2 domain‐binding motifs, indicating a potentially direct interaction with Csk. The interactions of Csk with four proteins (partitioning defective 3 (Par3), DDR1, SYK and protein kinase C iota) were confirmed using biochemical approaches and phosphotyrosine 1127 of Par3 C‐terminus was proved to directly bind to Csk‐SH2 domain, which was consistent with predictions from in silico analysis. Finally, immunofluorescence experiments revealed co‐localization of Csk with Par3 in tight junction (TJ) in a tyrosine phosphorylation‐dependent manner and overexpression of Csk, but not its SH2‐domain mutant lacking binding to phosphotyrosine, promoted the TJ assembly in Madin‐Darby canine kidney cells, implying the involvement of Csk‐SH2 domain in regulating cellular TJs. In conclusion, the newly identified potential interacting partners of Csk provided new insights into its functional diversity in regulation of numerous cellular events, in addition to controlling the SFK activity.     

The folding pathway of a functionally competent C-terminal domain of nucleophosmin: Protein stability and denatured state residual structure

Nucleophosmin (NPM1) is a nucleolar protein implicated in ribosome biogenesis, centrosome duplication and cell cycle control; the NPM1 gene is the most frequent target for mutations in Acute Myeloid Leukemia. Mutations map to the C-terminal domain of the protein and cause its unfolding, loss of DNA binding properties and aberrant cellular localization. Here we investigate the folding pathway and denatured state properties of a NPM1 C-terminal domain construct encompassing the last 70 residues in the reference sequence. This construct is more stable than the previously characterized domain, which consisted of the last 53 residues. Data reveal that, similarly to what was discovered for the shorter construct, also the 70-residue construct of NPM1 displays a detectable residual structure in its denatured state. The higher stability of the latter domain allows us to conclude that the denatured state is robust to changes in solvent composition and that it consists of a discrete state in equilibrium with the expanded fully unfolded conformation. This observation, which might appear as a technicality, is in fact of general importance for the understanding of the folding of proteins. The implications of our results are discussed in the context of previous works on single domain helical proteins.     

Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation.

The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, delta Gu (H2O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing delta Gu (H2O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed.     

Kinetic nonoptimality and vibrational stability of proteins

Scaling of folding times in Go models of proteins and of decoy structures with the Lennard-Jones potentials in the native contacts reveal power law trends when studied under optimal folding conditions. The power law exponent depends on the type of native geometry. Its value indicates lack of kinetic optimality in the model proteins. In proteins, mechanical and thermodynamic stabilities are correlated.     

Opsin stability and folding: modulation by phospholipid bicelles

Integral membrane proteins do not fare well when extracted from biological membranes and are unstable or lose activity in detergents commonly used for structure and function investigations. We show that phospholipid bicelles provide a valuable means of preserving alpha-helical membrane proteins in vitro by supplying a soluble lipid bilayer fragment. Both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-[(cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (Chaps) and DMPC/l-α-1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles dramatically increase the stability of the mammalian vision receptor rhodopsin as well as its apoprotein, opsin. Opsin is particularly unstable in detergent solution but can be directly purified into DMPC/Chaps. We show that opsin can also be directly purified in DMPC/DHPC bicelles to give correctly folded functional opsin, as shown by the ability to regenerate rhodopsin to  70% yield. These well-characterised DMPC/DHPC bicelles enable us to probe the influence of bicelle properties on opsin stability. These bicelles are thought to provide DMPC bilayer fragments with most DHPC capping the bilayer edge, giving a soluble bilayer disc. Opsin stability is shown to be modulated by the q value, the ratio of DMPC to DHPC, which reflects changes in the bicelle size and, thus, proportion of DMPC bilayer present. The observed changes in stability also correlate with loss of opsin secondary structure as determined by synchrotron far-UV circular dichroism spectroscopy; the most stable bicelle results in the least helix loss. The inclusion of Chaps rather than DHPC in the DMPC/Chaps bicelles, however, imparts the greatest stability. This suggests that it is not just the DMPC bilayer fragment in the bicelles that stabilises the protein, but that Chaps provides additional stability either through direct interaction with the protein or by altering the DMPC/Chaps bilayer properties within the bicelle. The significant stability enhancements and preservation of secondary structure reported here in bicelles are pertinent to other membrane proteins, notably G-protein-coupled receptors, which are unstable in detergent solution.     

A novel procedure for identification of the folding/unfolding patterns of dimeric proteins

The unfolding of proteins has been widely used for investigating the thermodynamic properties of monomeric proteins but has been used infrequently for dimeric (or oligomeric) proteins, because of the inherent cooperation of denaturation and dissociation of the dimers (oligomers). Here, we introduce a thermodynamic parameter K_obs to discriminate the diverse folding patterns of dimeric proteins. K_obs remains constant as the protein concentration increases for the true one-step curve of unfolding pattern (A), increases and reaches a plateau for one-step curves with monomeric intermediate pattern (B), and increases steadily with no plateau for one-step curves with dimeric intermediate pattern (C).     

Tagging of Escherichia coli proteins with new cassettes allowing in vivo systematic fluorescent and luminescent detection,and purification from physiological expression levels

We designed cassettes allowing the systematic fusion of fluorescent or luminescent proteins preceded by the calmodulin binding peptide tag to the C–terminus of Escherichia coli proteins. The chromosomal insertion, and thus physiological expression level of these fusions, permits the study of protein localization by fluorescent microscopy and protein quantification, in vivo and dynamically in diverse conditions. Furthermore, the calmodulin binding peptide tag allows standard detection, affinity purification, and co–purification experiments. These cassettes are therefore very valuable for the versatility of experiments they make available for a given strain, from biochemistry to dynamic and in vivo studies.     

Alpha-hairpin stability and folding of transmembrane segments

Molecular Dynamics (MD) simulations at low dielectric constant have been carried out for peptides matching the double spanning segments of transmembrane proteins. Different folding dynamics have been observed. The peptides folded into the stable helix-turn-helix conformation-alpha-hairpin-with antiparallel-oriented strands or unstable alpha-hairpin conformation that unfolded later into the straight helical structure. The peptide having flexible residues in the TM helices often misfolded into a tangled structure that can be avoided by restricting the flexibility of these residues. General conclusions can be drawn from the observed folding dynamics. The stability and folding of some double spanning transmembrane fragments are self-assembling. The following and/or neighboring peptide chains of the protein may support the stability of the hairpin structure of other fragments. The stability of the TM helices containing flexible residues could be maintained due to contacts with neighboring TM segments.