Conformational dynamics in the disordered region of human CPEB3 linked to memory consolidation.

BACKGROUND: Current understanding of the molecular basis of memory consolidation points to an important function of amyloid formation by neuronal-specific isoforms of the cytoplasmic polyadenylation element binding (CPEB) protein family. In particular, CPEB is thought to promote memory persistence through formation of self-sustaining prion-like amyloid assemblies at synapses, mediated by its intrinsically disordered region (IDR) and leading to permanent physical alterations at the basis of memory persistence. Although the molecular mechanisms by which amyloid formation takes place in CPEB have been described in invertebrates, the way amyloid formation occurs in the human homolog CPEB3 (hCPEB3) remains unclear. Here, we characterize by NMR spectroscopy the atomic level conformation and ps-ms dynamics of the 426-residue IDR of hCPEB3, which has been associated with episodic memory in humans. RESULTS: We show that the 426-residue N-terminal region of hCPEB3 is a dynamic, intrinsically disordered region (IDR) which lacks stable folded structures. The first 29 residues, M1QDDLLMDKSKTQPQPQQQQRQQQQPQP29, adopt a helical + disordered motif, and residues 86-93: P83QQPPPP93, and 166-175: P166PPPAPAPQP175 form polyproline II (PPII) helices. The (VG)5 repeat motif is completely disordered, and residues 200-250 adopt three partially populated α-helices. Residues 345-355, which comprise the nuclear localization signal (NLS), form a modestly populated α-helix which may mediate STAT5B binding. These findings allow us to suggest a model for nascent hCPEB3 structural transitions at single residue resolution, advancing that amyloid breaker residues, like proline, are a key difference between functional versus pathological amyloids. CONCLUSION: Our NMR spectroscopic analysis of hCPEB3 provides insights into the first structural transitions involved in protein-protein and protein-mRNA interactions. The atomic level understanding of these structural transitions involved in hCPEB3 aggregation is a key first step toward understanding memory persistence in humans, as well as sequence features that differentiate beneficial amyloids from pathological ones. AREAS: Biophysics, Structural Biology, Biochemistry & Neurosciences.

Glycine rich segments adopt polyproline II helices: Implications for biomolecular condensate formation.

Many intrinsically disordered proteins contain Gly-rich regions which are generally assumed to be disordered. Such regions often form biomolecular condensates which play essential roles in organizing cellular processes. However, the bases of their formation and stability are still not completely understood. Based on NMR studies of the Gly-rich H. harveyi "snow flea" antifreeze protein, we recently proposed that Gly-rich sequences, such as the third "RGG" region of Fused in Sarcoma (FUS) protein, may adopt polyproline II helices whose association might stabilize condensates. Here, this hypothesis is tested with a polypeptide corresponding to the third RGG region of FUS. NMR spectroscopy and molecular dynamics simulations suggest that significant populations of polyproline II helix are present. These findings are corroborated in a model peptide Ac-RGGYGGRGGWGGRGGY-NH2, where a peak characteristic of polyproline II helix is observed using CD spectroscopy. Its intensity suggests a polyproline II population of 40%. This result is supported by data from FTIR and NMR spectroscopies. In the latter, NOE correlations are observed between the Tyr and Arg, and Arg and Trp side chain hydrogens, confirming that side chains spaced three residues apart are close in space. Taken together, the data are consistent with a polyproline II helix, which is bent to optimize interactions between guanidinium and aromatic moieties, in equilibrium with a statistical coil ensemble. These results lend credence to the hypothesis that Gly-rich segments of disordered proteins may form polyproline II helices which help stabilize biomolecular condensates.

The structural determinants that lead to the formation of particular oligomeric structures in the pancreatic-type ribonuclease family.

Pancreatic-type ribonucleases are a family of RNA degrading enzymes that share different degrees of sequence identity but a very similar 3D-structure. The prototype of this family is bovine pancreatic ribonuclease or ribonuclease A. This enzyme has been the object of landmark work on the folding, stability, protein chemistry, catalysis, enzyme-substrate interaction and molecular evolution. In the recent years, the interest in the study of pancreatic-type ribonucleases has increased due to the involvement of some members of this family in special biological functions. In addition, dimeric and also higher oligomeric structures can be attained by the members of this family. The oligomers described structurally to date are mainly formed by 3D-domain swapping, a process which consists of the exchange of identical domains (i.e. identical structural elements, usually the N- and C-termini) between the subunits and is considered to be a mechanism for amyloid-type aggregate formation. This review compares the dimeric and oligomeric structures of different members of the pancreatic-type ribonuclease family which are able to acquire these structures, namely, bovine seminal ribonuclease, ribonuclease A and its human counterpart, human pancreatic ribonuclease. A specific focus is placed on what is known about the structural determinants that lead to the acquisition of a particular oligomeric structure and on the proposed mechanism of 3D-swapping.

The solution structure and dynamics of human pancreatic ribonuclease determined by NMR spectroscopy provide insight into its remarkable biological activities and inhibition.

Human pancreatic ribonuclease (RNase 1) is expressed in many tissues; has several important enzymatic and biological activities, including efficient cleavage of single-stranded RNA, double-stranded RNA and double-stranded RNA-DNA hybrids, digestion of dietary RNA, regulation of vascular homeostasis, inactivation of the HIV, activation of immature dendritic cells and induction of cytokine production; and furthermore shows potential as an anti-tumor agent. The solution structure and dynamics of uncomplexed, wild-type RNase 1 have been determined by NMR spectroscopy methods to better understand these activities. The family of 20 structures determined on the basis of 6115 unambiguous nuclear Overhauser enhancements is well resolved (pairwise backbone RMSD=1.07 A) and has the classic RNase A type of tertiary structure. Important structural differences compared with previously determined crystal structures of RNase 1 variants or inhibitor-bound complexes are observed in the conformation of loop regions and side chains implicated in the enzymatic as well as biological activities and binding to the cytoplasmic RNase inhibitor. Multiple side chain conformations observed for key surface residues are proposed to be crucial for membrane binding as well as translocation and efficient RNA hydrolysis. (15)N-(1)H relaxation measurements interpreted with the standard and our extended Lipari-Szabo formalism reveal rigid regions and identify more dynamic loop regions. Some of the most dynamic areas are key for binding to the cytoplasmic RNase inhibitor. This finding and the important differences observed between the structure in solution and that bound to the inhibitor are indications that RNase 1 to inhibitor binding can be better described by the "induced fit" model rather than the rigid "lock-into-key" mechanism. Translational diffusion measurements reveal that RNase 1 is predominantly dimeric above 1 mM concentration; the possible implications of this dimeric state for the remarkable biological properties of RNase 1 are discussed.

Pressure-jump-induced kinetics reveals a hydration dependent folding/unfolding mechanism of ribonuclease A.

Pressure-jump (p-jump)-induced relaxation kinetics was used to explore the energy landscape of protein folding/unfolding of Y115W, a fluorescent variant of ribonuclease A. Pressure-jumps of 40 MPa amplitude (5 ms dead-time) were conducted both to higher (unfolding) and to lower (folding) pressure, in the range from 100 to 500 MPa, between 30 and 50 degrees C. Significant deviations from the expected symmetrical protein relaxation kinetics were observed. Whereas downward p-jumps resulted always in single exponential kinetics, the kinetics induced by upward p-jumps were biphasic in the low pressure range and monophasic at higher pressures. The relative amplitude of the slow phase decreased as a function of both pressure and temperature. At 50 degrees C, only the fast phase remained. These results can be interpreted within the framework of a two-dimensional energy surface containing a pressure- and temperature-dependent barrier between two unfolded states differing in the isomeric state of the Asn-113-Pro-114 bond. Analysis of the activation volume of the fast kinetic phase revealed a temperature-dependent shift of the unfolding transition state to a larger volume. The observed compensation of this effect by glycerol offers an explanation for its protein stabilizing effect.

Some biological actions of PEG-conjugated RNase A oligomers.

Previously we have shown that monomeric RNase A has no significant biological activity, whereas its oligomers (dimer to tetramer) prepared by lyophilizing from 50% acetic acid solutions, show remarkable aspermatogenic and antitumor activities. Furthermore, conjugates prepared by chemical binding of native RNase A to polyethylene glycol (PEG) have shown a significant aspermatogenic and antitumor activities. In this work we show that the chemical conjugation of PEG to the RNase A C-dimer, and to the two RNase A trimers (NC-trimer and C- trimer) decreases the aspermatogenic activity of the oligomers while increasing their inhibitory activity on the growth of the human UB900518 amelanotic melanoma transplanted in athymic nude mice. Moreover, the PEG-conjugated RNaseA oligomers are devoid, like the free oligomers, of any embryotoxic activity.

Solution structure and dynamics of ribonuclease Sa.

We have used NMR methods to characterize the structure and dynamics of ribonuclease Sa in solution. The solution structure of RNase Sa was obtained using the distance constraints provided by 2,276 NOEs and the C6-C96 disulfide bond. The 40 resulting structures are well determined; their mean pairwise RMSD is 0.76 A (backbone) and 1.26 A (heavy atoms). The solution structures are similar to previously determined crystal structures, especially in the secondary structure, but exhibit new features: the loop composed of Pro 45 to Ser 48 adopts distinct conformations and the rings of tyrosines 51, 52, and 55 have reduced flipping rates. Amide protons with greatly reduced exchange rates are found predominantly in interior beta-strands and the alpha-helix, but also in the external 3/10 helix and edge beta-strand linked by the disulfide bond. Analysis of (15)N relaxation experiments (R1, R2, and NOE) at 600 MHz revealed five segments, consisting of residues 1-5, 28-31, 46-50, 60-65, 74-77, retaining flexibility in solution. The change in conformation entropy for RNase SA folding is smaller than previously believed, since the native protein is more flexible in solution than in a crystal.

The cadherin cytoplasmic domain is unstructured in the absence of beta-catenin. A possible mechanism for regulating cadherin turnover.

Cadherins are single pass transmembrane proteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion by linking the cytoskeletons of adjacent cells. In adherens junctions, the cytoplasmic domain of cadherins bind to beta-catenin, which in turn binds to the actin-associated protein alpha-catenin. The physical properties of the E-cadherin cytoplasmic domain and its interactions with beta-catenin have been investigated. Proteolytic sensitivity, tryptophan fluorescence, circular dichroism, and (1)H NMR measurements indicate that murine E-cadherin cytoplasmic domain is unstructured. Upon binding to beta-catenin, the domain becomes resistant to proteolysis, suggesting that it structures upon binding. Cadherin-beta-catenin complex stability is modestly dependent on ionic strength, indicating that, contrary to previous proposals, the interaction is not dominated by electrostatics. Comparison of 18 cadherin sequences indicates that their cytoplasmic domains are unlikely to be structured in isolation. This analysis also reveals the presence of PEST sequences, motifs associated with ubiquitin/proteosome degradation, that overlap the previously identified beta-catenin-binding site. It is proposed that binding of cadherins to beta-catenin prevents recognition of degradation signals that are exposed in the unstructured cadherin cytoplasmic domain, favoring a cell surface population of catenin-bound cadherins capable of participating in cell adhesion.

Folding kinetics of phage 434 Cro protein.

Folding kinetics for phage 434 Cro protein are examined and compared with those reported for lambda(6-85), the N-terminal domain of the repressor of phage lambda. The two proteins have similar all-helical structures consisting of five helices but different stabilities. In contrast to lambda(6-85), sharp and distinct aromatic (1)H NMR signals without exchange broadening characterize the native and urea-denatured 434 Cro forms at equilibrium at 20 degrees C, indicating slow interconversion on the NMR time scale. Stopped-flow fluorescence data using the single 434 Cro tryptophan indicate strongly urea-dependent refolding rates and smaller urea dependencies of the unfolding rates, suggesting a native-like transition state ensemble. Refolding rates are slower and unfolding rates considerably faster at pH 4 than at pH 6. This accounts for the lower stability of 434 Cro at pH 4 and suggests the existence of pH-dependent, possibly salt bridge interactions that are more stabilizing at pH 6. At <2 M urea, decreased folding amplitudes and nonlinear urea dependencies that are apparent at pH 6 indicate deviation from two-state behavior and suggest the formation of an early folding intermediate. The folding behavior of 434 Cro and why it folds 2 orders of magnitude slower than lambda(6-85) are rationalized in terms of the lower intrinsic helix stabilities and putative charge interactions in 434 Cro.

Thermodynamic analysis of the structural stability of phage 434 Cro protein.

Thermodynamic parameters describing the phage 434 Cro protein have been determined by calorimetry and, independently, by far-UV circular dichroism (CD) measurements of isothermal urea denaturations and thermal denaturations at fixed urea concentrations. These equilibrium unfolding transitions are adequately described by the two-state model. The far-UV CD denaturation data yield average temperature-independent values of 0.99 +/- 0.10 kcal mol(-)(1) M(-)(1) for m and 0.98 +/- 0.05 kcal mol(-)(1) K(-)(1) for DeltaC(p)()(,U), the heat capacity change accompanying unfolding. Calorimetric data yield a temperature-independent DeltaC(p)()(,U) of 0.95 +/- 0.30 kcal mol(-)(1) K(-)(1) or a temperature-dependent value of 1.00 +/- 0.10 kcal mol(-)(1) K(-)(1) at 25 degrees C. DeltaC(p)()(,U) and m determined for 434 Cro are in accord with values predicted using known empirical correlations with structure. The free energy of unfolding is pH-dependent, and the protein is completely unfolded at pH 2.0 and 25 degrees C as judged by calorimetry or CD. The stability of 434 Cro is lower than those observed for the structurally similar N-terminal domain of the repressor of phage 434 (R1-69) or of phage lambda (lambda(6)(-)(85)), but is close to the value reported for the putative monomeric lambda Cro. Since a protein's structural stability is important in determining its intracellular stability and turnover, the stability of Cro relative to the repressor could be a key component of the regulatory circuit controlling the levels and, consequently, the functions of the two proteins in vivo.

Helix-stabilizing nonpolar interactions between tyrosine and leucine in aqueous and TFE solutions: 2D-1H NMR and CD studies in alanine-lysine peptides.

Interactions between side chains spaced (i,i + 3) and (i,i + 4) may explain the context dependence of helix propensities observed in different systems. Nonpolar residues with these spacings occur frequently in protein helices and stabilize isolated peptide helices. Here (i,i + 3) and (i,i + 4) nonpolar interactions between Tyr and Leu in different solution conditions are studied in detail in alanine-based peptides using 2D 1H NMR and CD spectroscopy. Helix contents analyzed using current models for helix-coil transitions yield interaction energies which demonstrate significant helix stabilization in aqueous 1 M NaCl solutions by Tyr-Leu or Leu-Tyr pairs when spaced (i,i + 4) and, to a smaller extent, when spaced (i,i + 3), comparable to those estimated for other residue pairs. The interactions persist in solutions containing TFE, a helix-stabilizing solvent believed to diminish hydrophobic interactions, but not in helix-destabilizing 6 M urea. 1H NMR resonances for all peptides and solution conditions except in 6 M urea were completely assigned. NMR data indicate that the N-terminal residues are more helical and that the N-acetyl group participates in helix formation. The two (i,i + 4) spaced pairs show the same pattern of NOE cross-peaks between the Tyr and Leu side chains, as do the two (i,i + 3) pairs in 1 M NaCl as well in TFE solutions, and correspond well with that expected for the specific Tyr-Leu pair with side-chain contacts in protein helices.

A pulse-chase-competition experiment to determine if a folding intermediate is on or off-pathway: application to ribonuclease A.

A modified pulse-chase experiment is applied to determine if the native-like intermediate IN of ribonuclease A is on or off-pathway. The 1H label retained in the native protein is compared when separate samples of 1H-labeled IN and unfolded protein are allowed to fold to native in identical conditions. The solvent is 2H2O and the pH* is such that the unfolded protein rapidly exchanges its peptide NH protons with solvent, and IN does not. If IN is on-pathway, more 1H-label will be retained in the test sample starting with IN than in the control sample starting with unfolded protein. The results show that IN is a productive (on-pathway) intermediate. Application of the modified pulse-chase experiment to the study of rapidly formed folding intermediates may be possible when a rapid mixing device is used.

Protein folding: matching theory and experiment.

The impact of folding funnels and folding simulations on the way experimentalists interpret results is examined. The image of the transition state has changed from a unique species that has a strained configuration, with a correspondingly high free energy, to a more ordinary folding intermediate, whose balance between limited conformational entropy and stabilizing contacts places it at the top of the free energy barrier. Evidence for a broad transition barrier comes from studies showing that mutations can change the position of the barrier. The main controversial issue now is whether populated folding intermediates are productive on-pathway intermediates or dead-end traps. Direct experimental evidence is needed. Theories suggesting that populated intermediates are trapped in a glasslike state are usually based on mechanisms which imply that trapping would only be extremely short-lived (e.g., nanoseconds) in water at 25 degrees C. There seems to be little experimental evidence for long-lived trapping in monomers, if folding aggregates are excluded. On the other hand, there is good evidence for kinetic trapping in dimers. alpha-Helix formation is currently the fastest known process in protein folding, and incipient helices are present at the start of folding. Fast helix formation has the effect of narrowing drastically the choice of folding routes. Thus helix formation can direct folding. It changes the folding metaphor from pouring liquid down a folding funnel to a train leaving a switchyard with only a few choices of exit tracks.

Characterization of the unfolding pathway of hen egg white lysozyme.

After the recent discovery of a ribonuclease A unfolding intermediate [Kiefhaber, T., et al. (1995) Nature 375, 513-515], we investigated the unfolding pathway of hen egg white lysozyme. At pH* 4.00 with D2O at 10 degrees C and 6 M guanidinium chloride, unfolding shows a single, slow kinetic phase, with a relaxation time of 3300 s when monitored by circular dichroism (CD). Exchange of the tryptophan indole nitrogen protons shows that buried Trp residues 123, 111, and 108 lose tight packing and become solvent-exposed simultaneously, with a mean relaxation time of 3300 s, similar to the CD-monitored unfolding rate. Unfolding monitored by Trp fluorescence shows, moreover, that 90% of the amplitude change occurs in a slow phase, with a relaxation time of 2400 s. Faster-unfolding phases with minor amplitudes are detected by Trp indole hydrogen exchange and by fluorescence. It is likely that these changes are caused by Trp 62 and Trp 63, active site residues which are not buried in the hydrophobic core. Lysozyme unfolding was further monitored by the histidine 15 C epsilon1 proton, which gives resolved lines for the native and unfolded species in one-dimensional 1H-NMR spectra. The majority of the unfolding reaction, 70%, occurs in a slow phase with a relaxation time of 3600 s, but there is also a rapid unfolding phase; 30% of the His 15 C epsilon1 proton resonance intensity is found at the unfolded chemical shift within tens of seconds after the start of unfolding. The amplitude of the rapid unfolding phase increases proportionally with the concentration of GdmCl denaturant present. These results show that a partially buried residue of lysozyme, histidine 15, takes part in forming an unfolding intermediate similar to the one observed earlier for valine 63 in ribonuclease A. The tryptophan side chains buried in the hydrophobic core of lysoyzme, in contrast, do not participate in forming the unfolding intermediate, as judged by proton chemical shifts. The buried tryptophan residues of dihydrofolate reductase, monitored by 19F-NMR, do participate in forming an unfolding intermediate [Hoeltzli, S. D., & Frieden, C. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9318-9322]; the difference between that study and ours may reside in the greater sensitivity of 19F to the detection of motional differences.

Different protein sequences can give rise to highly similar folds through different stabilizing interactions.

We report an interesting case of structural similarity between 2 small, nonhomologous proteins, the third domain of ovomucoid (ovomucoid) and the C-terminal fragment of ribosomal L7/L12 protein (CTF). The region of similarity consists of a 3-stranded beta-sheet and an alpha-helix. This region is highly similar; the corresponding elements of secondary structure share a common topology, and the RMS difference for "equivalent" C alpha atoms is 1.6 A. Surprisingly, this common structure arises from completely different sequences. For the common core, the sequence identity is less than 3%, and there is neither significant sequence similarity nor similarity in the position or orientation of conserved hydrophobic residues. This superposition raises the question of how 2 entirely different sequences can produce an identical structure. Analyzing this common region in ovomucoid revealed that it is stabilized by disulfide bonds. In contrast, the corresponding structure in CTF is stabilized in the alpha-helix by a composition of residues with high helix-forming propensities. This result suggests that different sequences and different stabilizing interactions can produce an identical structure.

Structural similarity of DNA-binding domains of bacteriophage repressors and the globin core.

BACKGROUND: In recent years, the determination of large numbers of protein structures has created a need for automatic and objective methods for the comparison of structures or conformations. Many protein structures show similarities of conformation that are undetectable by comparing their sequences. Comparison of structures can reveal similarities between proteins thought to be unrelated, providing new insight into the interrelationships of sequence, structure and function. RESULTS: Using a new tool that we have developed to perform rapid structural alignment, we present the highlights of an exhaustive comparison of all pairs of protein structures in the Brookhaven protein database. Notably, we find that the DNA-binding domain of the bacteriophage repressor family is almost completely embedded in the larger eight-helix fold of the globin family of proteins. The significant match of specific residues is correlated with functional, structural and evolutionary information. CONCLUSION: Our method can help to identify structurally similar folds rapidly and with high-sensitivity, providing a powerful tool for analyzing the ever-increasing number of protein structures being elucidated.

Urea denaturation of barnase: pH dependence and characterization of the unfolded state.

To investigate the pH dependence of the conformational stability of barnase, urea denaturation curves were determined over the pH range 2-10. The maximum conformational stability of barnase is 9 kcal mol-1 and occurs between pH 5 and 6. The dependence of delta G on urea concentration increases from 1850 cal mol-1 M-1 at high pH to about 3000 cal mol-1 M-1 near pH 3. This suggests that the unfolded conformations of barnase become more accessible to urea as the net charge on the molecule increases. Previous studies suggested that in 8 M urea barnase unfolds more completely than ribonuclease T1, even with the disulfide bonds broken [Pace, C.N., Laurents, D. V., & Thomson, J.A. (1990) Biochemistry 29, 2564-2572]. In support of this, solvent perturbation difference spectroscopy showed that in 8 M urea the Trp and Tyr residues in barnase are more accessible to perturbation by dimethyl sulfoxide than in ribonuclease T1 with the disulfide bonds broken.

Purification of recombinant ribonuclease T1 expressed in Escherichia coli.

A protocol for the rapid purification of ribonuclease T1 expressed from a chemically synthesized gene cloned into Escherichia coli is described. QAE ion-exchange and Sephadex G-50 chromatography are used to give over 300 mg (88% yield) of pure ribonuclease T1 from 61 of liquid culture in 3 days. We also report a new absorption coefficient for RNase T1: E1%278 nm = 15.4.

pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1.

To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein.