Kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis

The amyloidogenic homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to partially denatured monomers in order to aggregate. TTR contains two distinct quaternary interfaces, one of which defines the binding sites for thyroxine and small-molecule amyloidogenesis inhibitors. Kinetic stabilization of the tetramer can be accomplished either by the binding of amyloidogenesis inhibitors selectively to the native state over the dissociative transition state or by the introduction of trans-suppressor subunits (T119M) into heterotetramers to destabilize the dissociative transition state. In each case, increasing the dissociation activation barrier prevents tetramer dissociation. Herein, we demonstrate that tethering two subunits whose quaternary interface defines the thyroxine binding site also dramatically increases the barrier for tetramer dissociation, apparently by destabilization of the dissociative transition state. The tethered construct (TTR-L-TTR)2 is structurally and functionally equivalent to wild-type TTR. Urea is unable to denature (TTR-L-TTR)2, yet it is able to maintain the denatured state once denaturation is achieved by GdnHCl treatment, suggesting that (TTR-L-TTR)2 is kinetically rather than thermodynamically stabilized, consistent with the identical wild-type TTR and (TTR-L-TTR)2 GdnHCl denaturation curves. Studies focused on a construct containing a single TTR-L-TTR chain and two normal monomer subunits establish that alteration of only one quaternary structural interface is sufficient to impose kinetic stabilization on the entire quaternary structure.     

Effects of co-operative ligand binding on protein amide NH hydrogen exchange

Amide protection factors have been determined from NMR measurements of hydrogen/deuterium amide NH exchange rates measured on assigned signals from Lactobacillus casei apo-DHFR and its binary and ternary complexes with trimethoprim (TMP), folinic acid and coenzymes (NADPH/NADP(+)). The substantial sizes of the residue-specific DeltaH and TDeltaS values for the opening/closing events in NH exchange for most of the measurable residues in apo-DHFR indicate that sub-global or global rather than local exchange mechanisms are usually involved. The amide groups of residues in helices and sheets are those most protected in apo-DHFR and its complexes, and the protection factors are generally related to the tightness of ligand binding. The effects of ligand binding that lead to changes in amide protection are not localised to specific binding sites but are spread throughout the structure via a network of intramolecular interactions. Although the increase in protein stability in the DHFR.TMP.NADPH complex involves increased ordering in the protein structure (requiring TDeltaS energy) this is recovered, to a large extent, by the stronger binding (enthalpic DeltaH) interactions made possible by the reduced motion in the protein. The ligand-induced protection effects in the ternary complexes DHFR.TMP.NADPH (large positive binding co-operativity) and DHFR.folinic acid.NADPH (large negative binding co-operativity) mirror the co-operative effects seen in the ligand binding. For the DHFR.TMP.NADPH complex, the ligand-induced protection factors result in DeltaDeltaG(o) values for many residues being larger than the DeltaDeltaG(o) values in the corresponding binary complexes. In contrast, for DHFR.folinic acid.NADPH, the DeltaDeltaG(o) values are generally smaller than many of those in the corresponding binary complexes. The results indicate that changes in protein conformational flexibility on formation of the ligand complex play an important role in determining the co-operativity in the ligand binding.     

Hydration and packing are crucial to amyloidogenesis as revealed by pressure studies on transthyretin variants that either protect or worsen amyloid disease

The formation of amyloid aggregates is the hallmark of the amyloidogenic diseases. Transthyretin (TTR) is involved in senile systemic amyloidosis (wild-type protein) and familial amyloidotic polyneuropathy (point mutants). Through the use of high hydrostatic pressure (HHP), we compare the stability among wild-type (wt) TTR, two disease-associated mutations (V30M and L55P) and a trans-suppressor mutation (T119M). Our data show that the amyloidogenic conformation, easily populated in the disease-associated mutant L55P, can be induced by a cycle of compression-decompression with the wt protein rendering the latter highly amyloidogenic. After decompression, the recovered wt structure has weaker subunit interactions (loosened tetramer, T(4)(*)) and presents a stability similar to L55P, suggesting that HHP induces a defective fold in the wt protein, converting it to an altered conformation already present in the aggressive mutant, L55P. On the other hand, glucose, a chemical chaperone, can mimic the trans-suppression mutation by stabilizing the native state and by decreasing the amyloidogenic potential of the wt TTR at pH 5.0. The sequence of pressure stability observed was: L55P相似文献   

The solid-state catalytic isotope exchange of hydrogen in dalargin

A [³H]Dalargin preparation with a molar radioactivity of 52 Ci/mmol was obtained by the high temperature solid-state catalytic isotope exchange (HSCIE) of tritium for hydrogen at 150°C. This tritium-labeled peptide was shown to completely retain its biological activity in the test of binding to opioid receptors from rat brain. The dissociation constant of the Dalargin-opioid receptor complex was found to be 4.3 nM. The dependences of the chemical yield and the molar radioactivity on the reaction time and temperature of HSCIE were determined. The activation energy of the HSCIE reaction for the peptide was calculated to be 32 kcal/mol. The amino acid analysis showed that tritium is distributed between all the amino acid residues of [³H]Dalargin at the HSCIE reaction, with the temperature growth significantly increasing the total tritium incorporation and, especially, enhancing the radioactivity incorporation into aromatic residues.     

Equilibrium hydrogen exchange reveals extensive hydrogen bonded secondary structure in the on-pathway intermediate of Im7

The four-helical immunity protein Im7 folds through an on-pathway intermediate that has a specific, but partially misfolded, hydrophobic core. In order to gain further insight into the structure of this species, we have identified the backbone hydrogen bonds formed in the ensemble by measuring the amide exchange rates (under EX2 conditions) of the wild-type protein and a variant, I72V. In this mutant the intermediate is significantly destabilised relative to the unfolded state (deltadeltaG(ui) = 4.4 kJ/mol) but the native state is only slightly destabilised (deltadeltaG(nu) = 1.8 kJ/mol) at 10 degrees C in 2H2O, pH* 7.0 containing 0.4 M Na2SO4, consistent with the view that this residue forms significant non-native stabilising interactions in the intermediate state. Comparison of the hydrogen exchange rates of the two proteins, therefore, enables the state from which hydrogen exchange occurs to be identified. The data show that amides in helices I, II and IV in both proteins exchange slowly with a free energy similar to that associated with global unfolding, suggesting that these helices form highly protected hydrogen-bonded helical structure in the intermediate. By contrast, amides in helix III exchange rapidly in both proteins. Importantly, the rate of exchange of amides in helix III are slowed substantially in the Im7* variant, I72V, compared with the wild-type protein, whilst other amides exchange more rapidly in the mutant protein, in accord with the kinetics of folding/unfolding measured using chevron analysis. These data demonstrate, therefore, that local fluctuations do not dominate the exchange mechanism and confirm that helix III does not form stable secondary structure in the intermediate. By combining these results with previously obtained Phi-values, we show that the on-pathway folding intermediate of Im7 contains extensive, stable hydrogen-bonded structure in helices I, II and IV, and that this structure is stabilised by both native and non-native interactions involving amino acid side-chains in these helices.     

High hydrostatic pressure dissociates early aggregates of TTR105-115, but not the mature amyloid fibrils

A range of disorders such as Alzheimer's disease and type II diabetes have been linked to protein misfolding and aggregation. Transthyretin is an amyloidogenic protein which is involved in familial amyloid polyneuropathy, the most common form of systemic amyloid disease. A peptide fragment of this protein, TTR105-115, has been shown to form well-defined amyloid fibrils in vitro. In this study, the stability of amyloid fibrils towards high hydrostatic pressure has been investigated by Fourier transform infrared spectroscopy. Information on the morphology of the species exposed to high hydrostatic pressure was obtained by atomic force microscopy. The species formed early in the aggregation process were found to be dissociated by relatively low hydrostatic pressure (220 MPa), whereas mature fibrils are pressure insensitive up to 1.3 GPa. The pressure stability of the mature fibrils is consistent with a fibril structure in which there is an extensive hydrogen bond network in a tightly packed environment from which water is excluded. The fact that early aggregates can be dissociated by low pressure suggests, however, that hydrophobic and electrostatic interactions are the dominant factors stabilizing the species formed in the early stages of fibril formation.     

Equilibrium amide hydrogen exchange and protein folding kinetics

The classical Linderstrøm-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (IUN). On the other hand, in an on-pathway three-state system (UIN), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments.     

Differences in hydrogen exchange behavior between the oxidized and reduced forms of Escherichia coli thioredoxin.

Amide proton exchange of thioredoxin is used to monitor the structural effects of reduction of its single disulfide. An effective 3-5-proton difference between the oxidized and reduced protein form is observed early in proton out-exchange of the whole protein, which is independent of temperature in the range of 5-45 degrees C, indicating that redox-sensitive changes are probably not due to low-energy structural fluctuations. Medium resolution hydrogen exchange experiments have localized the redox-sensitive amide protons to two parts of the sequence that are distant from each other in the three-dimensional structure: the active-site turn and the first beta-strand. The sum of the proton differences observed in the peptides from these regions is equal to that of the whole protein, indicating that all redox-sensitive hydrogen exchange effects are observed in the peptide experiments. A model combining structural changes within the protein matrix with changes in the surface hydration properties is proposed as a mechanism for the communication between distant sites within the protein. Sound velocity and density measurements of reduced and oxidized thioredoxin are presented in the accompanying paper (Kaminsky, S.M. & Richards, F.M., 1992, Protein Sci. 1, 22-30).     

Mapping the energy landscape of repeat proteins using NMR-detected hydrogen exchange

Repeat proteins contain tandem arrays of a simple structural motif. In contrast to globular proteins, repeat proteins are stabilized only by interactions between residues that are relatively close together in the sequence, with no ”long-range” interactions. Our work focuses on the tetratricopeptide repeat (TPR), a 34 amino acid helix-turn-helix motif found in tandem arrays in many natural proteins. Earlier, we reported the design and characterization of a series of consensus TPR (CTPR) proteins, which are built as arrays of multiple tandem copies of a 34 amino acid consensus sequence. Here, we present the results of extensive hydrogen exchange (HX) studies of the folding-unfolding behavior of two CTPR proteins (CTPR2 and CTPR3). We used HX to detect and characterize partially folded species that are populated at low frequency in the nominally folded state. We show that for both proteins the equilibrium folding-unfolding transition is non-two-state, but sequential, with the outermost helices showing a significantly higher probability than inner helices of being unfolded. We show that the experimentally observed unfolding behavior is consistent with the predictions of a simple Ising model, in which individual helices are treated as ”spin-equivalents”. The results that we present have general implications for our understanding of the thermodynamic properties of repeat proteins.     

Hydrogen-deuterium exchange strategy for delineation of contact sites in protein complexes

We use NMR spectra to determine protein-protein contact sites by observing differences in amide proton hydrogen-deuterium exchange in the complex compared to the free protein in solution. Aprotic organic solvents are used to preserve H/D labeling patterns that would be scrambled in water solutions. The binding site between the mammalian co-chaperone Aha1 with the middle domain of the chaperone Hsp90 obtained by our H/D exchange method corresponds well with that in the X-ray crystal structure of the homologous complex from yeast, even to the observation of a secondary binding site. This method can potentially provide data for complexes with unknown structure and for large or dynamic complexes inaccessible via NMR and X-ray methods.     

Domain study of bacteriophage p22 coat protein and characterization of the capsid lattice transformation by hydrogen/deuterium exchange

Viral capsids are dynamic structures which undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Limited proteolysis and biochemical experiments indicated that the coat protein consists of two domains connected by a flexible loop. To investigate the properties and roles of the sub-domains, we have cloned them and initiated structure and function studies. The N-terminal domain, which is made up of 190 amino acid residues, is largely unstructured in solution, while the C-terminal domain, which consists of 239 amino acid residues, forms a stable non-covalent dimer. The N-terminal domain adopts additional structure in the context of the C-terminal domain which might form a platform on which the N-terminal domain can fold. The local dynamics of the coat protein in both procapsids and mature capsids was monitored by hydrogen/deuterium exchange combined with mass spectrometry. The exchange rate for C-terminal domain peptides was similar in both forms. However, the N-terminal domain was more flexible in the empty procapsid shells than in the mature capsids. The flexibility of the N-terminal domain observed in the solution persisted into the procapsid form, but was lost upon maturation. The loop region connecting the two domains exchanged rapidly in the empty procapsid shells, but more slowly in the mature capsids. The global stabilization of the N-terminal domain and the flexibility encoded in the loop region may be a key component of the maturation process.     

Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

TTR (transthyretin) amyloidoses are diseases characterized by the aggregation and extracellular deposition of the normally soluble plasma protein TTR. Ex vivo and tissue culture studies suggest that tissue damage precedes TTR fibril deposition, indicating that early events in the amyloidogenic cascade have an impact on disease development. We used a human cardiomyocyte tissue culture model system to define these events. We previously described that the amyloidogenic V122I TTR variant is cytotoxic to human cardiac cells, whereas the naturally occurring, stable and non-amyloidogenic T119M TTR variant is not. We show that most of the V122I TTR interacting with the cells is extracellular and this interaction is mediated by a membrane protein(s). In contrast, most of the non-amyloidogenic T119M TTR associated with the cells is intracellular where it undergoes lysosomal degradation. The TTR internalization process is highly dependent on membrane cholesterol content. Using a fluorescent labelled V122I TTR variant that has the same aggregation and cytotoxic potential as the native V122I TTR, we determined that its association with human cardiomyocytes is saturable with a K_D near 650 nM. Only amyloidogenic V122I TTR compete with fluorescent V122I for cell-binding sites. Finally, incubation of the human cardiomyocytes with V122I TTR but not with T119M TTR, generates superoxide species and activates caspase 3/7. In summary, our results show that the interaction of the amyloidogenic V122I TTR is distinct from that of a non-amyloidogenic TTR variant and is characterized by its retention at the cell membrane, where it initiates the cytotoxic cascade.     

NMR investigations on residue level unfolding thermodynamics in DLC8 dimer by temperature dependent native state hydrogen exchange

Understanding protein stability at residue level detail in the native state ensemble of a protein is crucial to understanding its biological function. At the same time, deriving thermodynamic parameters using conventional spectroscopic and calorimetric techniques remains a major challenge for some proteins due to protein aggregation and irreversibility of denaturation at higher temperature values. In this regard, we describe here the NMR investigations on the conformational stabilities and related thermodynamic parameters such as local unfolding enthalpies, heat capacities and transition midpoints in DLC8 dimer, by using temperature dependent native state hydrogen exchange; this protein aggregates at high (>65°C) temperatures. The stability (free energy) of the native state was found to vary substantially with temperature at every residue. Significant differences were found in the thermodynamic parameters at individual residue sites indicating that the local environments in the protein structure would respond differently to external perturbations; this reflects on plasticity differences in different regions of the protein. Further, comparison of this data with similar data obtained from GdnHCl dependent native state hydrogen exchange indicated many similarities at residue level, suggesting that local unfolding transitions may be similar in both the cases. This has implications for the folding/unfolding mechanisms of the protein. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hydrogen exchange during cellulose synthesis distinguishes climatic and biochemical isotope fractionations in tree rings

The abundance of the hydrogen isotope deuterium (D) in tree rings is an attractive record of climate; however, use of this record has proved difficult so far, presumably because climatic and physiological influences on D abundance are difficult to distinguish. Using D labelling, we created a D gradient in trees. Leaf soluble sugars of relatively low D abundance entered cellulose synthesis in stems containing strongly D-labelled water. We used nuclear magnetic resonance (NMR) spectroscopy to quantify D in the C-H groups of leaf glucose and of tree-ring cellulose. Ratios of D abundances of individual C-H groups of leaf glucose depended only weakly on leaf D labelling, indicating that the D abundance pattern was determined by physiological influences. The D abundance pattern of tree-ring cellulose revealed C-H groups that exchanged strongly (C(2)-H) or weakly (C(6)-H2) with water during cellulose synthesis. We propose that strongly exchanging C-H groups of tree-ring cellulose adopt a climate signal stemming from the D abundance of source water. C-H groups that exchange weakly retain their D abundance established in leaf glucose, which reflects physiological influences. Combining both types of groups may allow simultaneous reconstruction of climate and physiology from tree rings.     

Equilibrium and kinetic folding of rabbit muscle triosephosphate isomerase by hydrogen exchange mass spectrometry

Unfolding and refolding of rabbit muscle triosephosphate isomerase (TIM), a model for (betaalpha)8-barrel proteins, has been studied by amide hydrogen exchange/mass spectrometry. Unfolding was studied by destabilizing the protein in guanidine hydrochloride (GdHCl) or urea, pulse-labeling with 2H2O and analyzing the intact protein by HPLC electrospray ionization mass spectrometry. Bimodal isotope patterns were found in the mass spectra of the labeled protein, indicating two-state unfolding behavior. Refolding experiments were performed by diluting solutions of TIM unfolded in GdHCl or urea and pulse-labeling with 2H2O at different times. Mass spectra of the intact protein labeled after one to two minutes had three envelopes of isotope peaks, indicating population of an intermediate. Kinetic modeling indicates that the stability of the folding intermediate in water is only 1.5 kcal/mol. Failure to detect the intermediate in the unfolding experiments was attributed to its low stability and the high concentrations of denaturant required for unfolding experiments. The folding status of each segment of the polypeptide backbone was determined from the deuterium levels found in peptic fragments of the labeled protein. Analysis of these spectra showed that the C-terminal half folds to form the intermediate, which then forms native TIM with folding of the N-terminal half. These results show that TIM folding fits the (4+4) model for folding of (betaalpha)8-barrel proteins. Results of a double-jump experiment indicate that proline isomerization does not contribute to the rate-limiting step in the folding of TIM.     

Biochemical characterisation of TCTP questions its function as a guanine nucleotide exchange factor for Rheb

Translationally controlled tumour protein (TCTP) is involved in malignant transformation and regulation of apoptosis. It has been postulated to serve as a guanine nucleotide exchange factor for the small G-protein Rheb. Rheb functions in the PI3 kinase/mTOR pathway. The study presented here was initiated to characterise the interaction between TCTP and Rheb biochemically. Since (i) no exchange activity of TCTP towards Rheb could be detected in vitro, (ii) no interaction between TCTP and Rheb could be detected by NMR spectroscopy, and (iii) no effect of TCTP depletion in cells on the direct downstream targets of Rheb could be observed in vivo, this study shows that TCTP is unlikely to be a guanine nucleotide exchange factor for Rheb.     

Solid phase isotope exchange with spillover hydrogen in amino acids,peptides, and proteins

We summarize here information on the theoretical and experimental study of high-temperature (150–200°C) solid phase catalytic isotope exchange (HSCIE) carried out with amino acids, peptides, and proteins under the action of spillover hydrogen. Main specific features of the HSCIE reaction, its mechanism, and its use for studying spatial interactions in polypeptides are discussed. A virtually complete absence of racemization makes this reaction a valuable preparative method. The main regularities of the HSCIE reaction with the participation of spillover tritium have been revealed in the case of peptides and proteins, and the dependence of reactivity of peptide fragments on the spatial organization of their molecules has been studied. An important peculiarity of this reaction is that HSCIE proceeds at 150–200°C with a high degree of chirality retention in amino acids and peptides. This is provided by its reaction mechanism, which consists in a synchronous one-center substitution at the saturated carbon atom characterized by the formation of pentacoordinated carbon and a three-center bond between the carbon and the incoming and outgoing hydrogen atoms.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 3–21.Original Russian Text Copyright © 2005 by Zolotarev, Dadayan, Borisov.     

The membrane-bound conformation of alpha-lactalbumin studied by NMR-monitored 1H exchange

The interaction of bovine alpha-lactalbumin (BLA) with negatively charged phospholipid bilayers was studied by NMR monitored 1H exchange to characterize the conformational transition that enables a water-soluble protein to associate with and partially insert into a membrane. BLA was allowed to exchange in deuterated buffer in the absence (reference) and the presence (membrane-bound) of acidic liposomes at pH 4.5, experimental conditions that allow efficient protein-membrane interaction. After adjusting the pH to 6.0, to dissociate the protein from the membrane, reference and membrane-released samples of BLA were analysed by (F1) band-selective homonuclear decoupled total correlation spectroscopy in the alphaH-NH region. The overall exchange behaviour of the membrane-bound state is molten globule-like, suggesting an overall destabilization of the polypeptide. Nevertheless, the backbone amide protons of residues R10, L12, C77, K94, K98, V99 and W104 show significant protection against solvent exchange in the membrane-bound protein. We propose a mechanism for the association of BLA with negatively charged membranes that includes initial protonation of acidic side-chains at the membrane interface, and formation of an interacting site with the membrane which involves helixes A and C. In the next step these helices would slide away from each other, adopting a parallel orientation to the membrane, and would rotate to maximize the interaction between their hydrophobic residues and the lipid bilayer.     

Characterisation of hydrogen bonding networks in RNAs via magic angle spinning solid state NMR spectroscopy

It is demonstrated that the spatial proximity of ¹H nuclei in hydrogen bonded base-pairs in RNAs can be conveniently mapped via magic angle spinning solid state NMR experiments involving proton spin diffusion driven chemical shift correlation of low gamma nuclei such as the imino and amino nitrogens of nucleic acid bases. As different canonical and non-canonical base-pairing schemes encountered in nucleic acids are characterised by topologically different networks of proton dipolar couplings, different base-pairing schemes lead to characteristic cross-peak intensity patterns in such correlation spectra. The method was employed in a study of a 100 kDa RNA composed of 97 CUG repeats, or (CUG)₉₇ that has been implicated in the neuromuscular disease myotonic dystrophy. ¹⁵N–¹⁵N chemical shift correlation studies confirm the presence of Watson–Crick GC base pairs in (CUG)₉₇.