A Simple Mechanism Underlying the Effect of Protecting Osmolytes on Protein Folding.

Osmolytes are small organic compounds that confer to the cell an enhanced adaptability to external conditions. Many osmolytes not only protect the cell from osmotic stress but also stabilize the native structure of proteins. While simplified models able to predict changes to protein stability are available, a general physicochemical explanation of the underlying microscopic mechanism is still missing. Here, we address this issue by performing very long all-atom MD simulations, free energy calculations, and experiments on a well-characterized mini-protein, the villin headpiece. Comparisons between the folding free energy landscapes in pure water and osmolyte solutions, together with experimental validation by means of circular dichroism, unfolding experiments, and NMR, led us to formulate a simple hypothesis for the protecting mechanism. Taken together, our results support a novel mechanistic explanation according to which the main driving force behind native state protection is a change in the solvent rotational diffusion.

Solution structure, backbone dynamics and chitin binding of the anti-fungal protein from Streptomyces tendae TU901.

AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae Tü901. Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases. No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g. agglutinins, lectins, hevein) is observed. AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown. Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy. The protein contains two antiparallel beta-sheets (five and four beta-strands each), that pack against each other in a parallel beta-sandwich. This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity. A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, beta-B2 and gamma-B crystallins from Bos taurus, canavalin from Jack bean). AFP1 is therefore classified as a new member of the betagamma-crystallin superfamily. The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data. We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15 degrees with that of the inertial tensor. After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein. Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1. The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction.

The highly refined solution structure of the cytotoxic ribonuclease alpha-sarcin reveals the structural requirements for substrate recognition and ribonucleolytic activity.

alpha-Sarcin selectively cleaves a single phosphodiester bond in a universally conserved sequence of the major rRNA, that inactivates the ribosome. The elucidation of the three-dimensional solution structure of this 150 residue enzyme is a crucial step towards understanding alpha-sarcin's conformational stability, ribonucleolytic activity, and its exceptionally high level of specificity. Here, the solution structure has been determined on the basis of 2658 conformationally relevant distances restraints (including stereoespecific assignments) and 119 torsional angular restraints, by nuclear magnetic resonance spectroscopy methods. A total of 60 converged structures have been computed using the program DYANA. The 47 best DYANA structures, following restrained energy minimization by GROMOS, represent the solution structure of alpha-sarcin. The resulting average pairwise root-mean-square-deviation is 0.86 A for backbone atoms and 1.47 A for all heavy atoms. When the more variable regions are excluded from the analysis, the pairwise root-mean-square deviation drops to 0.50 A and 1.00 A, for backbone and heavy atoms, respectively. The alpha-sarcin structure is similar to that reported for restrictocin, although some differences are clearly evident, especially in the loop regions. The average rmsd between the structurally aligned backbones of the 47 final alpha-sarcin structures and the crystal structure of restrictocin is 1.46 A. On the basis of a docking model constructed with alpha-sarcin solution structure and the crystal structure of a 29-nt RNA containing the sarcin/ricin domain, the regions in the protein that could interact specifically with the substrate have been identified. The structural elements that account for the specificity of RNA recognition are located in two separate regions of the protein. One is composed by residues 51 to 55 and loop 5, and the other region, located more than 11 A away in the structure, is the positively charged segment formed by residues 110 to 114.

Solution structure and dynamics of the Rous sarcoma virus capsid protein and comparison with capsid proteins of other retroviruses.

The solution structure and dynamics of the recombinant 240 amino acid residue capsid protein from the Rous sarcoma virus has been determined by NMR methods. The structure was determined using 2200 distance restraints and 330 torsion angle restraints, and the dynamics analysis was based on (15)N relaxation parameters (R(1), R(2), and (1)H-(15)N NOE) measured for 153 backbone amide groups. The monomeric protein consists of independently folded N- and C-terminal domains that comprise residues Leu14-Leu146 and Ala150-Gln226, respectively. The domains exhibit different rotational correlation times (16.6(+/-0.1) ns and 12.6(+/-0.1) ns, respectively), are connected by a flexible linker (Ala147-Pro149), and do not give rise to inter-domain NOE values, indicating that they are dynamically independent. Despite limited sequence similarity, the structure of the Rous sarcoma virus capsid protein is similar to the structures determined recently for the capsid proteins of retroviruses belonging to the lentivirus and human T-cell leukemia virus/bovine leukemia virus genera. Structural differences that exist in the C-terminal domain of Rous sarcoma virus capsid relative to the other capsid proteins appear to be related to the occurrence of conserved cysteine residues. Whereas most genera of retroviruses contain a pair of conserved and essential cysteine residues in the C-terminal domain that appear to function by forming an intramolecular disulfide bond during assembly, the Rous sarcoma virus capsid protein does not. Instead, the Rous sarcoma virus capsid protein contains a single cysteine residue that appears to be conserved among the avian C-type retroviruses and is positioned in a manner that might allow the formation of an intermolecular disulfide bond during capsid assembly.

Backbone dynamics of the N-terminal domain of the HIV-1 capsid protein and comparison with the G94D mutant conferring cyclosporin resistance/dependence.

Nuclear magnetic resonance (NMR) (15)N relaxation methods have been used to characterize the backbone dynamics of the N-terminal core domain of the HIV-1 capsid protein (CA(151)). The domain, which has an unusually flat, triangular shape, tumbles in solution at 28 degrees C with an effective rotational correlation time of 11.5 ns. Relaxation data for backbone amides in the domain's seven alpha-helices are indicative of fully anisotropic rotational diffusion. The principal axes of the rotational diffusion tensor calculated from the NMR data are aligned to within 12-23 degrees of the principal axes of the inertial tensor, with the axis of fastest rotational diffusion coincident with that of minimal inertia, and vice versa. Large variations in the (15)N-(1)H nuclear Overhauser effects for individual amino acids correlate with the degree of convergence in the previously calculated NMR structure. In particular, the partially disordered residues Val86-Arg97 that contain the human cyclophilin A (CypA) packaging signal have (15)N heteronuclear NOEs and transversal relaxation rates consistent with a high degree of dynamic conformational averaging. The N-terminal domain of a CA mutant (G94D) that confers both resistance to and dependence on cyclosporin A analogues was also analyzed. Our results indicate that this mutation does not influence the conformation or dynamics of CA(151), and therefore probably affects the function of the protein by modifying essential intermolecular CA-CA interactions.

Solution structure of the capsid protein from the human T-cell leukemia virus type-I.

The solution structure of the capsid protein (CA) from the human T-cell leukemia virus type one (HTLV-I), a retrovirus that causes T-cell leukemia and HTLV-I-associated myelopathy in humans, has been determined by NMR methods. The protein consists of independent N and C-terminal domains connected by a flexible linker. The domains are structurally similar to the N-terminal "core" and C-terminal "dimerization" domains, respectively, of the human immunodeficiency virus type one (HIV-1) and equine infectious anemia virus (EIAV) capsid proteins, although several important differences exist. In particular, hydrophobic residues near the major homology region are partially buried in HTLV-I CA, which is monomeric in solution, whereas analogous residues in HIV-1 and EIAV CA project from the C-terminal domain and promote dimerization. These differences in the structure and oligomerization state of the proteins appear to be related to, and possibly controlled by, the oxidation state of conserved cysteine residues, which are reduced in HTLV-I CA but form a disulfide bond in the HIV-1 and EIAV CA crystal structures. The results are consistent with an oxidative capsid assembly mechanism, in which CA oligomerization or maturation is triggered by disulfide bo nd formation as the budding virus enters the oxidizing environment of the bloodstream.

Characterization of pKa values and titration shifts in the cytotoxic ribonuclease alpha-sarcin by NMR. Relationship between electrostatic interactions, structure, and catalytic function.

The electrostatic behavior of titrating groups in alpha-sarcin was investigated using 1H NMR spectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in the molecule were determined over the pH range of 3.0-8.5. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the unambiguous determination of pKa values for all glutamic acid and histidine residues, as well as for the C-terminal carboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalytically relevant histidines, His50 and His137, and glutamic acid, Glu96, in the alpha-sarcin-2'-GMP complex were also determined. The pKa values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamic acids, and four histidines) were found to be close to their normal values. On the other hand, a number of side chain groups, including those in the active center, showed pKa values far from their intrinsic values. Thus, the pKa values for active site residues His50, Glu96, and His137 were 7.7, 5.2, and 5.8 in the free enzyme and 7.6, approximately 4.8, and 6.8 in the alpha-sarcin-2'-GMP complex, respectively. The pKa values and the activity profile against ApA, as a function of pH, are in agreement with the proposed enzymatic mechanism (in common with RNase T1 and the family of the microbial ribonucleases), in which Glu96 and His137 act as a general base and general acid, respectively. In almost all microbial ribonucleases, a Phe-His interaction is present, which affects the pKa of one of the His residues at the active site (His137). The absence of this interaction in alpha-sarcin would explain the lower pKa value of this His residue, and provides an explanation for the decreased RNase activity of this protein as compared to those of other microbial ribonucleases.

Characterisation of the isolated Che Y C-terminal fragment (79-129)--Exploring the structure/stability/folding relationship of the alpha/beta parallel protein Che Y.

To gain insight into how the three-dimensional structure, stability and folding of the protein Che Y are related to one another, we have performed a conformational analysis of a long fragment of this protein, encompassing its C-terminal 51 residues (79-129). This fragment consists of residues in the beta-strands 4 and 5 and alpha-helices 4 and 5 of native Che Y. The study has been performed by two-dimensional NMR and far-ultraviolet circular dichroism in aqueous solution and in 30% (by vol.) trifluoroethanol/ water at 273 K and 298 K. We observe little structure for this fragment in aqueous solution which could be due to low helical populations in the regions corresponding to helices 4 and 5. Within the limits of the residual helical structure experimentally detected, helix 4 appears to extend beyond the N-terminus observed in the native structure by over four residues belonging to the preceding loop. In 30% trifluoroethanol the helical content of both helices increase and helix 4 extends further to include the preceding beta-strand 4. None of the long-range NOEs present in native Che Y are observed under the explored experimental conditions. The conformational shifts of the H(alpha) protons within the alpha-helices of fragment 79-129 are identical to those of shorter synthetic peptides corresponding to the isolated alpha-helices. Thus, the fragment 79-129 appears to behave as an open chain with low local helical populations. The very low intrinsic ability for structure formation displayed by this region of Che Y at pH 2.5 suggests that in the folded protein this region could be mainly stabilised by interactions with the N-terminal Che Y region. This is in accordance with the contact map of Che Y, which shows that the strongest non-local contacts of C-terminal residues are with residues of the N-terminal region, while those within the C-terminal region are very weak. More importantly, the relationship appears to be possibly extended to the folding properties of the protein, since the C-terminal region is not structurally formed in the folding transition state of Che Y but in the final steps of the folding.

Structural basis for the catalytic mechanism and substrate specificity of the ribonuclease alpha-sarcin.

alpha-Sarcin is a ribosome-inactivating protein which selectively cleaves a single phosphodiester bond in a universally conserved sequence of the major rRNA. The solution structure of a-sarcin has been determined on the basis of 1898 distance and angular experimental constraints from NMR spectroscopy. It reveals a catalytic mechanism analogous to that of the T1 family of ribonucleases while its exquisite specificity resides in the contacts provided by its distinctive loops.

1H and 15N nuclear magnetic resonance assignment and secondary structure of the cytotoxic ribonuclease alpha-Sarcin.

The ribosome-inactivating protein alpha-Sarcin (alpha S) is a 150-residue fungal ribonuclease that, after entering sensitive cells, selectively cleaves a single phosphodiester bond in an universally conserved sequence of the major rRNA to inactivate the ribosome and thus exert its cytotoxic action. As a first step toward establishing the structure-dynamics-function relationships in this system, we have carried out the assignment of the 1H and 15N NMR spectrum of alpha S on the basis of homonuclear (1H-1H) and heteronuclear (1H-15N) two-dimensional correlation spectra of a uniformly 15N-labeled sample, and two selectively 15N-labeled (Tyr and Phe) samples, as well as a single three-dimensional experiment. The secondary structure of alpha S, as derived from the characteristic patterns of dipolar connectivities between backbone protons, conformational chemical shifts, and the protection of backbone amide protons against exchange, consists of a long N-terminal beta-hairpin, a short alpha-helical segment, and a C-terminal beta-sheet of five short strands arranged in a + 1, + 1, + 1, + 1 topology, connected by long loops in which the 13 Pro residues are located.

Characterization of the antifungal protein secreted by the mould Aspergillus giganteus.

An antifungal polypeptide (AFP) of 51 amino acid residues, secreted by the mould Aspergillus giganteus, has been purified to homogeneity and characterized. The inhibitory effect of this protein on the growth of different microorganisms has been studied. Whereas the growth of many of the filamentous fungi assayed is inhibited, no effect has been observed against yeasts or bacteria. The minimal concentration for total inhibition of the growth is in the range 6 to 25 microM. The antifungal polypeptide does not produce any effect on the growth of the producing mould. The polypeptide promotes aggregation of acidic phospholipid vesicles. A remarkable resistance to proteolysis and a low hydrogen x deuterium exchange have been observed for this protein. The protein does not show any thermal transition up to 80 degrees C when studied by differential scanning calorimetry and infrared spectroscopy. The uv absorbance, fluorescence emission, and circular dichroism (CD) characteristics of this protein have been studied. The protein exhibits a strong positive band at 230 nm as a prominent feature of the CD spectrum in the far uv region. All the spectroscopical properties of the antifungal protein are highly influenced by the abundance of tyrosine residues. These can be grouped in two different populations, buried and exposed, based on the results of pH-titration experiments. Fourier-transform infrared spectroscopy reveals a high content of beta-structure in AFP. Reduction and carboxy-amidomethylation produces a rather unstructured polypeptide as deduced from its spectroscopical properties.

NMR solution structure of the antifungal protein from Aspergillus giganteus: evidence for cysteine pairing isomerism.

The solution structure of the antifungal protein (AFP, 51 residues, 4 disulfide bridges) from Aspergillus giganteus has been determined by using experimentally derived interproton distance constraints from nuclear magnetic resonance (NMR) spectroscopy. Complete sequence-specific proton assignments were obtained at pH 5.0 and 35 degrees C. A set of 834 upper limit distance constraints from nuclear Overhauser effect measurements was used as input for the calculation of structures with the program DIANA. An initial family of 40 structures calculated with no disulfide constraints was used to obtain information about the disulfide connectivities, which could not be determined by standard biochemical methods. Three possible disulfide patterns were selected and the corresponding disulfide constraints applied to generate a family of 20 DIANA conformers for each pattern. Following energy minimization, the average pairwise RMSD of the 20 conformers of each family is 1.01, 0.89, and 1.01 A for backbone atoms and 1.82, 1.74, and 1.81 A for all heavy atoms. One of these three families contains the disulfide bridge arrangement actually present in the solution structure of AFP. Although the three families fulfill the NMR constraints, one of the disulfide patterns considered (cysteine pairs 7-33, 14-40, 26-49, 28-51) is favored among the others on the basis of previous chemical studies. It thus probably corresponds to the actual pattern of disulfide bridges present in the protein, and the corresponding family represents the solution structure of AFP. The folding of AFP consists of five antiparallel beta strands connected in a -1, -1, +3, +1 topology and highly twisted, defining a small and compact beta barrel stabilized by four internal disulfide bridges. A cationic site formed by up to three lysine side chains adjacent to a hydrophobic stretch, both at the protein surface, may constitute a potential binding site for phospholipids which would be the basis of its biological function. On the other hand, a second, minor form of AFP has been detected. NMR data, together with results from mass spectrometry, chemical analysis, and sedimentation equilibrium, suggest that this species differs from the major form in the pairs of cysteines involved in the four disulfide bridges.