Data requirements for reliable chemical shift assignments in deuterated proteins.

The information required for chemical shift assignments in large deuterated proteins was investigated using a Monte Carlo approach (Hitchens et al., 2002). In particular, the consequences of missing amide resonances on the reliability of assignments derived from C alpha and CO or from C alpha and C beta chemical shifts was investigated. Missing amide resonances reduce both the number of correct assignments as well as the confidence in these assignments. More significantly, a number of undetectable errors can arise when as few as 9% of the amide resonances are missing from the spectra. However, the use of information from residue specific labeling as well as local and long-range distance constraints improves the reliability and extent of assignment. It is also shown that missing residues have only a minor effect on the assignment of protein-ligand complexes using C alpha and CO chemical shifts and C alpha inter-residue connectivity, provided that the known chemical shifts of the unliganded protein are utilized in the assignment process.</p

Data requirements for reliable chemical shift assignments in deuterated proteins.

The information required for chemical shift assignments in large deuterated proteins was investigated using a Monte Carlo approach (Hitchens et al., 2002). In particular, the consequences of missing amide resonances on the reliability of assignments derived from C alpha and CO or from C alpha and C beta chemical shifts was investigated. Missing amide resonances reduce both the number of correct assignments as well as the confidence in these assignments. More significantly, a number of undetectable errors can arise when as few as 9% of the amide resonances are missing from the spectra. However, the use of information from residue specific labeling as well as local and long-range distance constraints improves the reliability and extent of assignment. It is also shown that missing residues have only a minor effect on the assignment of protein-ligand complexes using C alpha and CO chemical shifts and C alpha inter-residue connectivity, provided that the known chemical shifts of the unliganded protein are utilized in the assignment process.</p

MONTE: An automated Monte Carlo based approach to nuclear magnetic resonance assignment of proteins.

A general-purpose Monte Carlo assignment program has been developed to aid in the assignment of NMR resonances from proteins. By virtue of its flexible data requirements the program is capable of obtaining assignments of both heavily deuterated and fully protonated proteins. A wide variety of source data, such as inter-residue scalar connectivity, inter-residue dipolar (NOE) connectivity, and residue specific information, can be utilized in the assignment process. The program can also use known assignments from one form of a protein to facilitate the assignment of another form of the protein. This attribute is useful for assigning protein-ligand complexes when the assignments of the unliganded protein are known. The program can be also be used as an interactive research tool to assist in the choice of additional experimental data to facilitate completion of assignments. The assignment of a deuterated 45 kDa homodimeric Glutathione-S-transferase illustrates the principal features of the program.</p

Thermodynamic and Kinetic Analysis of Peptides Derived from CapZ, NDR, p53, HDM2, and HDM4 Binding to Human S100B

S100B is a member of the S100 subfamily of EF-hand proteins that has been implicated in malignant melanoma and neurodegenerative conditions such as Alzheimer’s disease and Parkinson’s disease. Calcium-induced conformational changes expose a hydrophobic binding cleft, facilitating interactions with a wide variety of nuclear, cytoplasmic, and extracellular target proteins. Previously, peptides derived from CapZ, p53, NDR, HDM2, and HDM4 have been shown to interact with S100B in a calcium-dependent manner. However, the thermodynamic and kinetic basis of these interactions remains largely unknown. To gain further insight, we screened these peptides against the S100B protein using isothermal titration calorimetry and nuclear magnetic resonance. All peptides were found to have binding affinities in the low micromolar to nanomolar range. Binding-induced changes in the line shapes of S100B backbone ¹H and ¹⁵N resonances were monitored to obtain the dissociation constants and the kinetic binding parameters. The large microscopic <i>K</i>_on rate constants observed in this study (≥1 × 10⁷ M^–1 s^–1) suggest that S100B utilizes a “fly casting mechanism” in the recognition of these peptide targets

NMR and Computation Reveal a Pressure-Sensitive Folded Conformation of Trp-Cage

Beyond defining the structure and stability of folded states of proteins, primary amino acid sequences determine all of the features of their conformational landscapes. Characterizing how sequence modulates the population of protein excited states or folding pathways requires atomic level detailed structural and energetic information. Such insight is essential for improving protein design strategies, as well as for interpreting protein evolution. Here, high pressure NMR and molecular dynamics simulations were combined to probe the conformational landscape of a small model protein, the tryptophan cage variant, Tc5b. Pressure effects on protein conformation are based on volume differences between states, providing a subtle continuous variable for perturbing conformations. 2D proton TOCSY spectra of Tc5b were acquired as a function of pressure at different temperature, pH, and urea concentration. In contrast to urea and pH which lead to unfolding of Tc5b, pressure resulted in modulation of the structures that are populated within the folded state basin. The results of molecular dynamics simulations on Tc5b displayed remarkable agreement with the NMR data. Principal component analysis identified two structural subensembles in the folded state basin, one of which was strongly destabilized by pressure. The pressure-dependent structural perturbations observed by NMR coincided precisely with the changes in secondary structure associated with the shifting populations in the folded state basin observed in the simulations. These results highlight the deep structural insight afforded by pressure perturbation in conjunction with high resolution experimental and advanced computational tools

Investigation into the Molecular and Thermodynamic Basis of Protein Interactions in Multimodal Chromatography Using Functionalized Nanoparticles

Although multimodal chromatography offers significant potential for bioseparations, there is a lack of molecular level understanding of the nature of protein binding in these systems. In this study a nanoparticle system is employed that can simulate a chromatographic resin surface while also being amenable to isothermal titration calorimetry (ITC) and solution NMR. ITC and NMR titration experiments are carried out with ¹⁵N-labeled ubiquitin to investigate the interactions of ubiquitin with nanoparticles functionalized with two industrially important multimodal ligands. The ITC results suggest that binding to both multimodal ligand surfaces is entropically driven over a range of temperatures and that this is due primarily to the release of surface bound waters. In order to reveal structural details of the interaction process, binding-induced chemical shift changes obtained from the NMR experiments are employed to obtain dissociation constants of individual amino acid residues on the protein surface. The residue level information obtained from NMR is then used to identify a preferred binding face on ubiquitin for interaction to both multimodal ligand surfaces. In addition, electrostatic potential and spatial aggregation propensity maps are used to determine important protein surface property data that are shown to correlate well with the molecular level information obtained from NMR. Importantly, the data demonstrate that the cluster of interacting residues on the protein surface act co-operatively to give rise to multimodal binding affinities several orders of magnitude greater than those obtained previously for interactions with free solution ligands. The use of NMR and ITC to study protein interactions with functionalized nanoparticles offers a new tool for obtaining important molecular and thermodynamic insights into protein affinity in multimodal chromatographic systems

High Pressure ZZ-Exchange NMR Reveals Key Features of Protein Folding Transition States

Understanding protein folding mechanisms and their sequence dependence requires the determination of residue-specific apparent kinetic rate constants for the folding and unfolding reactions. Conventional two-dimensional NMR, such as HSQC experiments, can provide residue-specific information for proteins. However, folding is generally too fast for such experiments. ZZ-exchange NMR spectroscopy allows determination of folding and unfolding rates on much faster time scales, yet even this regime is not fast enough for many protein folding reactions. The application of high hydrostatic pressure slows folding by orders of magnitude due to positive activation volumes for the folding reaction. We combined high pressure perturbation with ZZ-exchange spectroscopy on two autonomously folding protein domains derived from the ribosomal protein, L9. We obtained residue-specific apparent rates at 2500 bar for the N-terminal domain of L9 (NTL9), and rates at atmospheric pressure for a mutant of the C-terminal domain (CTL9) from pressure dependent ZZ-exchange measurements. Our results revealed that NTL9 folding is almost perfectly two-state, while small deviations from two-state behavior were observed for CTL9. Both domains exhibited large positive activation volumes for folding. The volumetric properties of these domains reveal that their transition states contain most of the internal solvent excluded voids that are found in the hydrophobic cores of the respective native states. These results demonstrate that by coupling it with high pressure, ZZ-exchange can be extended to investigate a large number of protein conformational transitions

Poly(ω-pentadecalactone)‑<i>b</i>‑poly(l‑lactide) Block Copolymers via Organic-Catalyzed Ring Opening Polymerization and Potential Applications

Poly­(pentadecalactone)-<i>b</i>-poly­(l-lactide) (PPDL-<i>b</i>-PLLA) diblock copolymers were prepared via the organic catalyzed ring-opening polymerization (ROP) of l-lactide (l-LA) from PPDL macroinitiators using either 1,8-diazabicyclo[5.4.0]­undec-7-ene (DBU) or 1,5,7-triazabicyclo[4.4.0]­dec-5-ene (TBD). Synthesis of PLLA blocks targeting degrees of polymerization (DP) up to 500 were found to yield diblock copolymers with crystalline PPDL and PLLA segments when TBD was used as the catalyst. The synthesis was further improved in a one-pot, two-step process using the same TBD catalyst for the synthesis of both segments. The application of these diblock copolymers as a compatibilizing agents resulted in homogenization of a biobased PLLA/poly­(ω-hydroxytetradecanoate) (90:10) blend upon a melt-process, yielding enhanced material properties

Polylactide/Poly(ω-hydroxytetradecanoic acid) Reactive Blending: A Green Renewable Approach to Improving Polylactide Properties

A green manufacturing technique, reactive extrusion (REx), was employed to improve the mechanical properties of polylactide (PLA). To achieve this goal, a fully biosourced PLA based polymer blend was conceived by incorporating small quantities of poly­(ω-hydroxytetradecanoic acid) (PC14). PLA/PC14 blends were compatibilized by transesterification reactions promoted by 200 ppm titanium tetrabutoxide (Ti­(OBu)₄) during REx. REx for 15 min at 150 rpm and 200 °C resulted in enhanced blend mechanical properties while minimizing losses in PLA molecular weight. SEM analysis of the resulting compatibilized phase-separated blends showed good adhesion between dispersed PC14 phases within the continuous PLA phase. Direct evidence for in situ synthesis of PLA-<i>b</i>-PC14 copolymers was obtained by HMBC and HSQC NMR experiments. The size of the dispersed phase was tuned by the screw speed to “tailor” the blend morphology. In the presence of 200 ppm Ti­(OBu)₄, inclusion of only 5% PC14 increased the elongation at break of PLA from 3 to 140% with only a slight decrease in the tensile modulus (3200 to 2900 MPa). Furthermore, PLA’s impact strength was increased by 2.4× that of neat PLA for 20% PC14 blends prepared by REx. Blends of PLA and PC14 are expected to expand the potential uses of PLA-based materials