Mechanical unfolding pathway and origin of mechanical stability of proteins of ubiquitin family: An investigation by steered molecular dynamics simulation

Like the muscle protein Titin, proteins of the ubiquitin family exhibit a parallel strand arrangement, but otherwise having a distinctly different fold and not involved in an obvious load‐bearing function, exhibit high resistance to mechanical unfolding. We have applied all‐atom molecular dynamics simulation technique in implicit solvent to present a deep insight into the force‐induced unfolding pathway of three proteins—ubiquitin, NEDD8, and SUMO‐2—all having almost similar structural features. Two intermediates evolve in the unfolding pathway of each of the three proteins. The first intermediate, which has already been identified in case of ubiquitin by earlier simulation results, is similar for ubiquitin and NEDD8, but different in SUMO‐2. We have found a new intermediate with β3–β4 hairpin and some residual α‐helical character; and this intermediate is common for all the three proteins. Thus, proteins of the ubiquitin family pass through a well‐defined conformation in their force‐induced unfolding pathway. Reason behind the higher mechanical stability of the proteins with parallel strand structures like Titin has also been identified. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Identification of a α‐helical molten globule intermediate and structural characterization of β‐cardiotoxin,an all β‐sheet protein isolated from the venom of Ophiophagus hannah (king cobra)

β‐Cardiotoxin is a novel member of the snake venom three‐finger toxin (3FTX) family. This is the first exogenous protein to antagonize β‐adrenergic receptors and thereby causing reduction in heart rates (bradycardia) when administered into animals, unlike the conventional cardiotoxins as reported earlier. 3FTXs are stable all β‐sheet peptides with 60–80 amino acid residues. Here, we describe the three‐dimensional crystal structure of β‐cardiotoxin together with the identification of a molten globule intermediate in the unfolding pathway of this protein. In spite of the overall structural similarity of this protein with conventional cardiotoxins, there are notable differences observed at the loop region and in the charge distribution on the surface, which are known to be critical for cytolytic activity of cardiotoxins. The molten globule intermediate state present in the thermal unfolding pathway of β‐cardiotoxin was however not observed during the chemical denaturation of the protein. Interestingly, circular dichroism (CD) and NMR studies revealed the presence of α‐helical secondary structure in the molten globule intermediate. These results point to substantial conformational plasticity of β‐cardiotoxin, which might aid the protein in responding to the sometimes conflicting demands of structure, stability, and function during its biological lifetime.     

The presence of non‐native helical structure in the unfolding of a beta‐sheet protein MPT63

MPT63, a major secreted protein from Mycobacterium tuberculosis, has been shown to have immunogenic properties and has been implicated in virulence. MPT63 is a β‐sandwich protein containing 11 β strands and a very short stretch of 3₁₀ helix. The detailed experimental and computational study reported here investigates the equilibrium unfolding transition of MPT63. It is shown that in spite of being a complete β‐sheet protein, MPT63 has a strong propensity toward helix structures in its early intermediates. Far UV‐CD and FTIR spectra clearly suggest that the low‐pH intermediate of MTP63 has enhanced helical content, while fluorescence correlation spectroscopy suggests a significant contraction. Molecular dynamics simulation complements the experimental results indicating that the unfolded state of MPT63 traverses through intermediate forms with increased helical characteristics. It is found that this early intermediate contains exposed hydrophobic surface, and is aggregation prone. Although MPT63 is a complete β‐sheet protein in its native form, the present findings suggest that the secondary structure preferences of the local interactions in early folding pathway may not always follow the native conformation. Furthermore, the Gly25Ala mutant supports the proposed hypothesis by increasing the non‐native helical propensity of the protein structure.     

C‐terminal deletion of leucine‐rich repeats from YopM reveals a heterogeneous distribution of stability in a cooperatively folded protein

Terminal deletions of units from α‐helical repeat proteins have provided insight into the physical origins of their cooperativity. To test if the same principles governing cooperativity apply to β‐sheet‐containing repeat proteins, we have created a series of C‐terminal deletion constructs from a large leucine‐rich repeat (LRR) protein, YopM. We have examined the structure and stability of the resulting deletion constructs by a combination of solution spectroscopy, equilibrium denaturation studies, and limited proteolysis. Surprisingly, a high degree of nonuniformity was found in the stability distribution of YopM. Unlike previously studied repeat proteins, we identified several key LRR that on deletion disrupt nearby structure, at distances as far away as up to three repeats, in YopM. This partial unfolding model is supported by limited proteolysis studies and by point substitution in repeats predicted to be disordered as a result of deletion of adjacent repeats. We show that key internal‐ and terminal‐caps must be present to maintain the structural integrity in adjacent regions (roughly four LRRs long) of decreased stability. The finding that full‐length YopM maintains a high level of cooperativity in equilibrium unfolding underscores the importance of interfacial interactions in stabilizing locally unstable regions of structure.     

Structural basis for unfolding pathway‐dependent stability of proteins: Vectorial unfolding versus global unfolding

Point mutations in proteins can have different effects on protein stability depending on the mechanism of unfolding. In the most interesting case of I27, the Ig‐like module of the muscle protein titin, one point mutation (Y9P) yields opposite effects on protein stability during denaturant‐induced “global unfolding” versus “vectorial unfolding” by mechanical pulling force or cellular unfolding systems. Here, we assessed the reason for the different effects of the Y9P mutation of I27 on the overall molecular stability and N‐terminal unraveling by NMR. We found that the Y9P mutation causes a conformational change that is transmitted through β‐sheet structures to reach the central hydrophobic core in the interior and alters its accessibility to bulk solvent, which leads to destabilization of the hydrophobic core. On the other hand, the Y9P mutation causes a bend in the backbone structure, which leads to the formation of a more stable N‐terminal structure probably through enhanced hydrophobic interactions.     

Temperature-dependent downhill unfolding of ubiquitin. II. Modeling the free energy surface

To provide evidence for the interpretation of temperature‐dependent unfolding kinetics and the downhill unfolding scenario presented in the accompanying experimental article (Part I), the free energy surface of ubiquitin unfolding is calculated using statistical mechanical models of the Muñoz‐Eaton (ME) form. The models allow only two states for each amino acid residue, folded or unfolded, and permutations of these states generate an ensemble of microstates. One‐dimensional free energy curves are calculated using the number of folded residues as a reaction coordinate. The proposed sequential unfolding of ubiquitin's β‐sheet is tested by mapping the free energy onto two reaction coordinates inspired by the experiment as follows: the number of folded residues in ubiquitin's stable β‐strands I and II and those of the less stable strands III–V. Although the original ME model successfully captures folding features of zipper‐like one‐dimensional folders, it misses important tertiary interactions between residues that are far from each other in primary sequence. To take tertiary contacts into account, partially folded microstates based on a spherical growth model are included in the calculation and compared with the original model. By calculating the folding probability of each residue for a given point on the free energy surface, the unfolding pathway of ubiquitin is visualized. At low temperature, thermal unfolding occurs along a sequential unfolding pathway as follows: disruption of the β‐strands III–V followed by unfolding of the strands I and II. At high temperature, multiple unfolding routes are formed. The heterogeneity of the transition state explains the global nonexponential unfolding observed in the T‐jump experiment at high temperature. The calculation also reports a high stability for the α‐helix of ubiquitin. Proteins 2008. © 2008 Wiley‐Liss, Inc.     

Differential stability of the bovine prion protein upon urea unfolding

Prion diseases, or transmissible spongiform encephalopathies, are a group of infectious neurological diseases associated with the structural conversion of an endogenous protein (PrP) in the central nervous system. There are two major forms of this protein: the native and noninfectious cellular form, PrP^C; and the misfolded, infectious, and proteinase K‐resistant form, PrP^Sc. The C‐terminal domain of PrP^C is mainly α‐helical in structure, whereas PrP^Sc in known to aggregate into an assembly of β‐sheets, forming amyloid fibrils. To identify the regions of PrP^C potentially involved in the initial steps of the conversion to the infectious conformation, we have used high‐resolution NMR spectroscopy to characterize the stability and structure of bovine recombinant PrP^C (residues 121 to 230) during unfolding with the denaturant urea. Analysis of the 800 MHz ¹H NMR spectra reveals region‐specific information about the structural changes occurring upon unfolding. Our data suggest that the dissociation of the native β‐sheet of PrP^C is a primary step in the urea‐induced unfolding process, while strong hydrophobic interactions between helices α1 and α3, and between α2 and α3, stabilize these regions even at very high concentrations of urea.     

Slow formation of aggregation‐resistant β‐sheet folding intermediates

Protein folding has been studied extensively for decades, yet our ability to predict how proteins reach their native state from a mechanistic perspective is still rudimentary at best, limiting our understanding of folding‐related processes in vivo and our ability to manipulate proteins in vitro. Here, we investigate the in vitro refolding mechanism of a large β‐helix protein, pertactin, which has an extended, elongated shape. At 55 kDa, this single domain, all‐β‐sheet protein allows detailed analysis of the formation of β‐sheet structure in larger proteins. Using a combination of fluorescence and far‐UV circular dichroism spectroscopy, we show that the pertactin β‐helix refolds remarkably slowly, with multiexponential kinetics. Surprisingly, despite the slow refolding rates, large size, and β‐sheet‐rich topology, pertactin refolding is reversible and not complicated by off‐pathway aggregation. The slow pertactin refolding rate is not limited by proline isomerization, and 30% of secondary structure formation occurs within the rate‐limiting step. Furthermore, site‐specific labeling experiments indicate that the β‐helix refolds in a multistep but concerted process involving the entire protein, rather than via initial formation of the stable core substructure observed in equilibrium titrations. Hence pertactin provides a valuable system for studying the refolding properties of larger, β‐sheet‐rich proteins, and raises intriguing questions regarding the prevention of aggregation during the prolonged population of partially folded, β‐sheet‐rich refolding intermediates. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Thermal unfolding pathway for the thermostable P22 tailspike endorhamnosidase

The conditions in which protein stability is biologically or industrially relevant frequently differ from those in which reversible denaturation is studied. The trimeric tailspike endorhamnosidase of phage P22 is a viral structural protein which exhibits high stability to heat, proteases, and detergents under a range of environmental conditions. Its intracellular folding pathway includes monomeric and trimeric folding intermediates and has been the subject of detailed genetic analysis. To understand the basis of tailspike thermostability, we have examined the kinetics of thermal and detergent unfolding. During thermal unfolding of the tailspike, a metastable unfolding intermediate accumulates which can be trapped in the cold or in the presence of SDS. This species is still trimeric, but has lost the ability to bind to virus capsids and, unlike the native trimer, is partially susceptible to protease digestion. Its N-terminal regions, containing about 110 residues, are unfolded whereas the central regions and the C-termini of the polypeptide chains are still in the folded state. Thus, the initiation step in thermal denaturation is the unfolding of the N-termini, but melting of the intermediate represents a second kinetic barrier in the denaturation process. This two-step unfolding is unusually slow at elevated temperature; for instance, in 2% SDS at 65 degrees C, the unfolding rate constant is 1.1 x 10(-3) s-1 for the transition from the native to the unfolding intermediate and 4.0 x 10(-5) s-1 for the transition from the intermediate to the unfolded chains. The sequential unfolding pathway explains the insensitivity of the apparent Tm to the presence of temperature-sensitive folding mutations [Sturtevant, J. M., Yu, M.-H., Haase-Pettingell, C., & King, J. (1989) J. Biol. Chem. 264, 10693-10698] which are located in the central region of the chain. The metastable unfolding intermediate has not been detected in the forward folding pathway occurring at lower temperatures. The early stage of the high-temperature thermal unfolding pathway is not the reverse of the late stage of the low-temperature folding pathway.     

Blockade of JAK2 activity suppressed accumulation of β‐catenin in leukemic cells

The Wnt/β‐catenin pathway has been implicated in leukemogenesis. We found β‐catenin abnormally accumulated in both human acute T cell leukemia Jurkat cells and human erythroleukemia HEL cells. β‐Catenin can be significantly down‐regulated by the Janus kinase 2 specific inhibitor AG490 in these two cells. AG490 also reduces the luciferase activity of a reporter plasmid driven by LEF/β‐catenin promoter. Similar results were observed in HEL cells infected with lentivirus containing shRNA against JAK2 gene. After treatment with 50 µM AG490 or shRNA, the mRNA expression levels of β‐catenin, APC, Axin, β‐Trcp, GSK3α, and GSK3β were up‐regulated within 12–16 h. However, only the protein levels of GSK3β and β‐Trcp were found to have increased relative to untreated cells. Knockdown experiments revealed that the AG490‐induced inhibition of β‐catenin can be attenuated by shRNA targeting β‐TrCP. Taken together; these results suggest that β‐Trcp plays a key role in the cross‐talk between JAK/STAT and Wnt/β‐catenin signaling in leukemia cells. J. Cell. Biochem. 111: 402–411, 2010. © 2010 Wiley‐Liss, Inc.     

BuildBeta—A system for automatically constructing beta sheets

We describe a method that can thoroughly sample a protein conformational space given the protein primary sequence of amino acids and secondary structure predictions. Specifically, we target proteins with β‐sheets because they are particularly challenging for ab initio protein structure prediction because of the complexity of sampling long‐range strand pairings. Using some basic packing principles, inverse kinematics (IK), and β‐pairing scores, this method creates all possible β‐sheet arrangements including those that have the correct packing of β‐strands. It uses the IK algorithms of ProteinShop to move α‐helices and β‐strands as rigid bodies by rotating the dihedral angles in the coil regions. Our results show that our approach produces structures that are within 4–6 Å RMSD of the native one regardless of the protein size and β‐sheet topology although this number may increase if the protein has long loops or complex α‐helical regions. Proteins 2010. © Published 2009 Wiley‐Liss, Inc.     

Role of electrostatic interactions for the stability and folding behavior of cold shock protein

The impacts of three charged‐residue‐involved mutations, E46A, R3E, and R3E/L66E, on the thermostability and folding behavior of the cold shock protein from the themophile Bacillus caldolyticus (Bc‐Csp) were investigated by using a modified Gō‐like model, in which the nonspecific electrostatic interactions of charged residues were taken into account. Our simulation results show that the wild‐type Bc‐Csp and its three mutants are all two‐sate folders, which is consistent with the experimental observations. It is found that these three mutations all lead to a decrease of protein thermodynamical stability, and the effect of R3E mutation is the strongest. The lower stability of these three mutants is due to the increase of the enthalpy of the folded state and the entropy of the unfolded state. Using this model, we also studied the folding kinetics and the folding/unfolding pathway of the wild‐type Bc‐Csp as well as its three mutants and then discussed the effects of electrostatic interactions on the folding kinetics. The results indicate that the substitutions at positions 3 and 46 largely decrease the folding kinetics, whereas the mutation of residue 66 only slightly decreases the folding rate. This result agrees well with the experimental observations. It is also found that these mutations have little effects on the folding transition state and the folding pathway, in which the N‐terminal β sheet folds earlier than the C‐terminal region. We also investigated the detailed unfolding pathway and found that it is really the reverse of the folding pathway, providing the validity of our simulation results. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

The acquisition of function is often associated with destabilizing mutations, giving rise to the stability–function tradeoff hypothesis. To test whether function is also accommodated at the expense of foldability, fibroblast growth factor‐1 (FGF‐1) was subjected to a comprehensive φ‐value analysis at each of the 11 turn regions. FGF‐1, a β‐trefoil fold, represents an excellent model system with which to evaluate the influence of function on foldability: because of its threefold symmetric structure, analysis of FGF‐1 allows for direct comparisons between symmetry‐related regions of the protein that are associated with function to those that are not; thus, a structural basis for regions of foldability can potentially be identified. The resulting φ‐value distribution of FGF‐1 is highly polarized, with the majority of positions described as either folded‐like or denatured‐like in the folding transition state. Regions important for folding are shown to be asymmetrically distributed within the protein architecture; furthermore, regions associated with function (i.e., heparin‐binding affinity and receptor‐binding affinity) are localized to regions of the protein that fold after barrier crossing (late in the folding pathway). These results provide experimental support for the foldability–function tradeoff hypothesis in the evolution of FGF‐1. Notably, the results identify the potential for folding redundancy in symmetric protein architecture with important implications for protein evolution and design.     

Telomere protection and TRF2 expression are enhanced by the canonical Wnt signalling pathway

The DNA‐binding protein TRF2 is essential for telomere protection and chromosome stability in mammals. We show here that TRF2 expression is activated by the Wnt/β‐catenin signalling pathway in human cancer and normal cells as well as in mouse intestinal tissues. Furthermore, β‐catenin binds to TRF2 gene regulatory regions that are functional in a luciferase transactivating assay. Reduced β‐catenin expression in cancer cells triggers a marked increase in telomere dysfunction, which can be reversed by TRF2 overexpression. We conclude that the Wnt/β‐catenin signalling pathway maintains a level of TRF2 critical for telomere protection. This is expected to have an important role during development, adult stem cell function and oncogenesis.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Protein prosthesis: β‐peptides as reverse‐turn surrogates

The introduction of non‐natural modules could provide unprecedented control over folding/unfolding behavior, conformational stability, and biological function of proteins. Success requires the interrogation of candidate modules in natural contexts. Here, expressed protein ligation is used to replace a reverse turn in bovine pancreatic ribonuclease (RNase A) with a synthetic β‐dipeptide: β²‐homoalanine–β³‐homoalanine. This segment is known to adopt an unnatural reverse‐turn conformation that contains a 10‐membered ring hydrogen bond, but one with a donor–acceptor pattern opposite to that in the 10‐membered rings of natural reverse turns. The RNase A variant has intact enzymatic activity, but unfolds more quickly and has diminished conformational stability relative to native RNase A. These data indicate that hydrogen‐bonding pattern merits careful consideration in the selection of beneficial reverse‐turn surrogates.     

Folding nucleus structure persists in thermally‐aggregated FGF‐1

An efficient protein‐folding pathway leading to target structure, and the avoidance of aggregation, is essential to protein evolution and de novo design; however, design details to achieve efficient folding and avoid aggregation are poorly understood. We report characterization of the thermally‐induced aggregate of fibroblast growth factor‐1 (FGF‐1), a small globular protein, by solid‐state NMR. NMR spectra are consistent with residual structure in the aggregate and provide evidence of a structured region that corresponds to the region of the folding nucleus. NMR data on aggregated FGF‐1 also indicate the presence of unstructured regions that exhibit hydration‐dependent dynamics and suggest that unstructured regions of aggregated FGF‐1 lie outside the folding nucleus. Since it is known that regions outside the folding nucleus fold late in the folding pathway, we postulate that these regions unfold early in the unfolding pathway and that the partially folded state is more prone to intermolecular aggregation. This interpretation is further supported by comparison with a designed protein that shares the same FGF‐1 folding nucleus sequence, but has different 1° structure outside the folding nucleus, and does not thermally aggregate. The results suggest that design of an efficient folding nucleus, and the avoidance of aggregation in the folding pathway, are potentially separable design criteria – the latter of which could principally focus upon the physicochemical properties of 1° structure outside the folding nucleus.     

Boosting protein stability with the computational design of β‐sheet surfaces

β‐sheets often have one face packed against the core of the protein and the other facing solvent. Mutational studies have indicated that the solvent‐facing residues can contribute significantly to protein stability, and that the preferred amino acid at each sequence position is dependent on the precise structure of the protein backbone and the identity of the neighboring amino acids. This suggests that the most advantageous methods for designing β‐sheet surfaces will be approaches that take into account the multiple energetic factors at play including side chain rotamer preferences, van der Waals forces, electrostatics, and desolvation effects. Here, we show that the protein design software Rosetta, which models these energetic factors, can be used to dramatically increase protein stability by optimizing interactions on the surfaces of small β‐sheet proteins. Two design variants of the β‐sandwich protein from tenascin were made with 7 and 14 mutations respectively on its β‐sheet surfaces. These changes raised the thermal midpoint for unfolding from 45°C to 64°C and 74°C. Additionally, we tested an empirical approach based on increasing the number of potential salt bridges on the surfaces of the β‐sheets. This was not a robust strategy for increasing stability, as three of the four variants tested were unfolded.