Human galectin-7 (Gal-7; also termed p53-induced gene 1 product) is a multifunctional effector by productive pairing with distinct glycoconjugates and protein counter-receptors in the cytoplasm and ...nucleus, as well as on the cell surface. Its structural analysis by NMR spectroscopy detected doubling of a set of particular resonances, an indicator of Gal-7 existing in two conformational states in slow exchange on the chemical shift time scale. Structural positioning of this set of amino acids around the P4 residue and loss of this phenomenon in the bioactive P4L mutant indicated cis-trans isomerization at this site. Respective resonance assignments confirmed our proposal of two Gal-7 conformers. Mapping hydrogen bonds and considering van der Waals interactions in molecular dynamics simulations revealed a structural difference for the N-terminal peptide, with the trans-state being more exposed to solvent and more mobile than the cis-state. Affinity for lactose or glycan-inhibitable neuroblastoma cell surface contact formation was not affected, because both conformers associated with an overall increase in order parameters (S2). At low µM concentrations, homodimer dissociation is more favored for the cis-state of the protein than its trans-state. These findings give direction to mapping binding sites for protein counter-receptors of Gal-7, such as Bcl-2, JNK1, p53 or Smad3, and to run functional assays at low concentration to test the hypothesis that this isomerization process provides a (patho)physiologically important molecular switch for Gal-7.
Galectins are a family of lectins with a conserved carbohydrate recognition domain that interacts with β-galactosides. By binding cell surface glycoconjugates, galectin-1 (gal-1) is involved in cell ...adhesion and migration processes and is an important regulator of tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate lactose binding to gal-1 and to derive solution NMR structures of gal-1 in the lactose-bound and unbound states. Structure analysis shows that the β-strands and loops around the lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that lactose binds the two carbohydrate recognition domains of the gal-1 dimer with negative cooperativity, in that the first lactose molecule binds more strongly (K1=21±6×103 M−1) than the second (K2=4±2×103 M−1). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K1=20±10×103 M−1 and K2=1.67±0.07×103 M−1. Molecular dynamics simulations provide insight into structural dynamics of the half-loaded lactose state and, together with NMR data, suggest that lactose binding at one site transmits a signal through the β-sandwich and loops to the second binding site. Overall, our results provide new insight into gal-1 structure–function relationships and to protein–carbohydrate interactions in general.
The product of p53-induced gene 1 is a member of the galectin family, i.e., galectin-7 (Gal-7). To move beyond structural data by X-ray diffraction, we initiated the study of the lectin by nuclear ...magnetic resonance (NMR) and circular dichroism spectroscopies, and molecular dynamics (MD) simulations. In concert, our results indicate that lactose binding to human Gal-7 induces long-range effects (minor conformational shifts and changes in structural dynamics) throughout the protein that result in stabilization of the dimer state, with evidence for positive cooperativity. Monte Carlo fits of (15)N-Gal-7 HSQC titrations with lactose using a two-site model yield K1 = 0.9 ± 0.6 × 10(3) M(-1) and K2 = 3.4 ± 0.8 × 10(3) M(-1). Ligand binding-induced stabilization of the Gal-7 dimer was supported by several lines of evidence: MD-based calculations of interaction energies between ligand-loaded and ligand-free states, gel filtration data and hetero-FRET spectroscopy that indicate a highly reduced tendency for dimer dissociation in the presence of lactose, CD-based thermal denaturation showing that the transition temperature of the lectin is significantly increased in the presence of lactose, and saturation transfer difference (STD) NMR using a molecular probe of the monomer state whose presence is diminished in the presence of lactose. MD simulations with the half-loaded ligand-bound state also provided insight into how allosteric signaling may occur. Overall, our results reveal long-range effects on Gal-7 structure and dynamics, which factor into entropic contributions to ligand binding and allow further comparisons with other members of the galectin family.
Protein stability is usually characterized calorimetrically by a melting temperature and related thermodynamic parameters. Despite its importance, the microscopic origin of the melting transition and ...the relationship between thermodynamic stability and dynamics remains a mystery. Here, NMR relaxation parameters were acquired for backbone
15NH groups of the 56 residue immunoglobulin-binding domain of streptococcal protein G over a pre-denaturation temperature range of 5–50
°C. Relaxation data were analyzed using three methods: the standard three-Lorentzian model free approach; the
F(ω)=2ω
J(ω) spectral density approach that yields motional correlation time distributions, and a new approach that determines frequency-dependent order parameters. Regardless of the method of analysis, the temperature dependence of internal motional correlation times and order parameters is essentially the same. Nanosecond time-scale internal motions are found for all NHs in the protein, and their temperature dependence yields activation energies ranging up to about 33
kJ/mol
residue. NH motional barrier heights are structurally correlated, with the largest energy barriers being found for residues in the most “rigid” segments of the fold: β-strands 1 and 4 and the α-helix. Trends in this landscape also parallel the free energy of folding–unfolding derived from hydrogen–deuterium (H–D) exchange measurements, indicating that the energetics for internal motions occurring on the nanosecond time-scale mirror those occurring on the much slower time-scale of H–D exchange. Residual heat capacities, derived from the temperature dependence of order parameters, range from near zero to near 100
J/mol
K
residue and correlate with this energy landscape. These results provide a unique picture of this protein's energy landscape and a relationship between thermodynamic stability and dynamics that suggests thermosensitive regions in the fold that could initiate the melting process.
Organized polymeric assemblies that incorporate bioactive sequences and structures are finding important applications for the study of protein structure−function relationships. We have recently ...described a heteropolymeric peptide−amphiphile system that forms organized structures in solution and on surfaces. While the overall three-dimensional features of peptide−amphiphiles have been studied previously, the precise environment of specific residues, particularly those within biologically active regions, have not been examined in detail. In the present study, we have used heteronuclear single quantum coherence (HSQC) and inverse-detected 1H−15N NMR spectroscopy to examine the structure and dynamics of a peptide and peptide−amphiphile that incorporate the α1(IV)1263−1277 (IV-H1) amino acid sequence from type IV collagen. Three variants of the sequence (Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4 were constructed with a single 15N-labeled Gly placed in the middle of the N-terminal (Gly-Pro-Hyp)4 region (residue Gly7), in the middle of the IV-H1 sequence (residue Gly19), or in the middle of the C-terminal (Gly-Pro-Hyp)4 region (residue Gly34). These peptides were also N-terminally acylated with hexanoic acid to create an analogous series of 15N-labeled peptide−amphiphiles. HSQC spectra indicated that both the peptide and the peptide−amphiphile were in triple-helical conformation at low temperature, supporting prior circular dichroism (CD) spectroscopic results. The intensities of the triple-helical cross-peaks were stronger for the peptide−amphiphile, consistent with an enhanced triple-helical thermal stability within the peptide−amphiphile construct compared to that of the peptide alone. Relative relaxation values for the peptide−amphiphile monomeric and trimeric species were consistent with those reported previously for other triple-helical peptides. Relaxation measurements indicated that the triple-helical IV-H1 region did not appear to be dramatically more flexible than the Gly-Pro-Hyp regions. The angle between Gly N−H bonds and the helix dyad axis, determined from the relaxation data, was within the range expected for triple helices. Overall, the peptide headgroup of the C6-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4 peptide−amphiphile appears to form a continuous triple helix that behaves similarly, in a dynamic sense, to a triple-helical peptide. The enhanced thermal stability of the peptide−amphiphile compared to the analogous triple-helical peptide, along with the multitude of organized structures formed by lipidlike compounds, suggest that peptide−amphiphiles could be utilized as targeted liposomes, sensors, receptors, or enzymes.
The model free approach, parameterized for the limiting case of isotropic molecular diffusion, is most often used to derive motional dynamics information from NMR relaxation data. However, many, if ...not most, biomolecules are asymmetric in shape and diffuse anisotropically in solution. Here, model free parameters have been derived to analyze NMR relaxation data for molecules undergoing anisotropic rotational diffusion. A simple correlation function has been formulated wherein restricted internal bond rotations are described by two parameters: 〈x 2 + y 2〉 and 〈x 2 − y 2〉. In the molecular frame, x and y are projections of motional unit vector a onto the plane that is perpendicular to the average position of vector a . For isotropic overall tumbling, this correlation function is reduced to a single parameter, 〈x 2 + y 2〉, which is related to the well-known model free order parameter as S 2 = 1−3 〈x 2 + y 2〉. The term 〈x 2 − y 2〉 becomes important and takes on a value for molecules tumbling anisotropically in solution. Various motional models are expressed in terms of 〈x 2 + y 2〉 and 〈x 2 - y 2〉, and the influence of rotational correlations, described by coefficients −1 < cij < 1, are considered. A simple protocol for using this new anisotropic motional model is provided, along with an example on a small α-helical peptide.
A novel approach is described to analyze NMR relaxation data on proteins. This method introduces the frequency-dependent order parameter,
S
2(
ω), in order to estimate contributions to the ...generalized order parameter
S
2 from different motional frequencies occurring on the picosecond to nanosecond time scales.
S
2(
ω) is defined as the sum of a specified set of weighting coefficients from the Lorentzian expansion of the spectral density function.
15
N
NMR relaxation data (500, 600, and 800
MHz) on protein GB1 exemplify the method. Using this approach provides information on motional restrictions over specific frequency or time scale ranges and provides a normalized comparison of motional restrictions between proteins having different overall tumbling correlation times.
Here, we report a method to simultaneously determine CH2 cross-correlation spectral densities and T1 relaxation times in the laboratory and rotating frames. To accomplish this, we have employed an ...indirect approach that is based on measurement of differences in relaxation rates acquired with and without cross-correlation terms. The new method, which can be employed using multidimensional NMR and standard relaxation pulse sequences, is validated experimentally by investigation of a selectively 13C-enriched hexadecapeptide and the uniformly 13C-enriched immunoglobulin-binding domain of streptococcal protein G (GB1). Use of this approach makes determination of CH2 cross-correlation spectral densities in uniformly 13C-enriched proteins now routine and provides novel information concerning their internal motions.
A new model-free approach that allows visualization of the distribution of motional correlation times is presented and is used to analyze 15N NMR relaxation data T 1, T 2, and {H}-15N NOE, acquired ...at 500, 600, and 800 MHz, on the uniformly 15N-enriched, 56 residue B1 domain from immunoglubulin protein G (GB1). Nanosecond time scale internal motions are found for all NHs of residues in protein GB1, a finding which is consistent with the concept of hierarchical internal motions in proteins first forwarded by Frauenfelder. Order parameters and overall tumbling correlation times derived using this approach are not influenced by the number of internal motional modes. Comparatively, use of the Lipari−Szabo, Clore et al., or LeMaster “model-free” methods yields underestimated order parameters and overestimated overall tumbling correlation times due to the presence of nanosecond time scale internal motions. This new model-free approach provides a straightforward way to derive more accurate order parameters and correlation times for overall tumbling and internal motions.
Many companies use additional personnel (group of divers) to determine the technical condition of propellers. This research will help to find the most effective method of machine learning, and the ...development on its basis will not only reduce the cost of technical diagnostics, but also accelerate the process itself due to autonomous underwater vehicle, thereby giving the opportunity to eliminate the need for human labor.
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