No description available.

No description available.

Understanding structure and folding of a protein is the key to understanding its biological function and potential role in diseases. Despite the importance of protein folding, a molecular level understanding of this process is still lacking. Solution-state nuclear magnetic resonance (NMR) is a powerful technique to investigate protein structure, dynamics, and folding mechanisms, since it provides residue-specific information. One of the major contributions that govern protein structure appears to be the interaction with the solvent. The importance of these interactions is particularly apparent in membrane proteins, which exist in an amphiphilic environment. Here, individual peptide fragments taken from the disulfide bond forming protein B (DsbB) were investigated in various solvents. The alpha-helical structures that were obtained, suggest that DsbB follows the two-stage model for folding. However, side chains of polar residues showed different conformations compared to the X-ray structure of fulllength protein, implying that polar side-chains may re-orient upon helix packing in order to form the necessary tertiary interactions that stabilize the global fold of DsbB. Model peptides in general represent attractive systems for the investigation of non-covalent interactions important for protein folding, including those with the solvent. NMR structures of the water soluble peptide, BBA5, were obtained in the presence an organic co-solvent, methanol. These structures indicate that the addition of methanol stabilizes an alpha-helix segment, but disrupts a hydrophobic cluster forming a beta-hairpin. Since dynamic effects reduce the ability for experimental observation of individual, bound solvent molecules, results were compared with molecular dynamics simulations. This comparison indicates that the observed effects of NMR structures are due to preferred binding of methanol and reduction of peptide-water hydrogen bonding. NMR structures, such as those determined here, represent a distribution of conformations under equilibrium. The dynamic process of protein unfolding can nevertheless be accessed through denaturation. A method was developed to probe thermal denaturation by measuring the temperature dependence of NOE intensity. Applied to a model peptide, trpzip4, it was confirmed that the beta-hairpin structure of this peptide is stabilized by the hydrophobic cluster formed by tryptophan residues. Together, the peptides investigated here illustrate the important roles that solvent-peptide interactions and side chain-side chain hydrophobic interactions play in forming stable secondary and tertiary structures.

No / Various host-guest peptide series are used by experimentalists as reference conformational states. One such use is as a baseline for random-coil NMR chemical shifts. Comparison to this random-coil baseline, through secondary chemical shifts, is used to infer protein secondary structure. The use of these random-coil data sets rests on the perception that the reference chemical shifts arise from states where there is little or no conformational bias. However, there is growing evidence that the conformational composition of natively and nonnatively unfolded proteins fail to approach anything that can be construed as random coil. Here, we use molecular dynamics simulations of an alanine-based host-guest peptide series (AAXAA) as a model of unfolded and denatured states to examine the intrinsic propensities of the amino acids. We produced ensembles that are in good agreement with the experimental NMR chemical shifts and confirm that the sampling of the 20 natural amino acids in this peptide series is be far from random. Preferences toward certain regions of conformational space were both present and dependent upon the environment when compared under conditions typically used to denature proteins, i.e., thermal and chemical denaturation. Moreover, the simulations allowed us to examine the conformational makeup of the underlying ensembles giving rise to the ensemble-averaged chemical shifts. We present these data as an intrinsic backbone propensity library that forms part of our Structural Library of Intrinsic Residue Propensities to inform model building, to aid in interpretation of experiment, and for structure prediction of natively and nonnatively unfolded states. / NIH GM 50789, Ministry of Education, Youth and Sports (MSMT) of the Czech Republic LH11020

Defining the intrinsic properties of amino acids which dictate the formation of helices, the most common protein secondary structure element, is an essential part of understanding protein folding. Pauling and co-workers initially predicted helical peptide folding motifs in the absence of solvent, suggesting that in vacuo studies may potentially discern the role of solvation in protein structure. Ion mobility-mass spectrometry (IMMS) combines a gas-phase ion separation based on collision cross-section (apparent surface area) with time-of-flight MS. The result is a correlation of collision cross-section with mass-to-charge, allowing detection of multiple conformations of the same ion. Most gas-phase peptide ions assume a compact, globular state that minimizes exposure to the low dielectric environment and maximizes intramolecular charge solvation. Conversely, a small number of peptides adopt a more extended (β-sheet or α-helix) conformation and exhibit a larger than predicted collision cross-section. Collision cross-sections measured using IM-MS are correlated with theoretical models generated using simulated annealing and allow for assignment of the overall ion structural motif (e.g. helix vs. chargesolvated globule). Here, two series of model peptides having known solution-phase helical propensities, namely Ac-(AAKAA)nY-NH2 (n = 3, 4, 5, 6 and 7) and Ac-Y(AEAAKA)nF-NH2 (n = 2, 3, 4, and 5), are investigated using IM-MS. Both protonated ([M + H]+) and metalcoordinated ([M + X]+ where X = Li, Na, K, Rb or Cs) species were analyzed to better understand the interplay of forces involved in gas-phase helical structure and stability. The data are analyzed using computational methods to examine the influence of peptide length, primary sequence, and number of basic (Lys, K) and acidic (Glu, E) residues on anhydrous ion structure.

No description available.

High-resolution magic angle spinning (HRMAS) NMR spectroscopy is used to study solid samples that are normally difficult to analyze due to broadening of peaks. Solid-phase peptide synthesis can bind peptides to an insoluble resin that can be analyzed with HRMAS NMR spectroscopy. A combination of HRMAS NMR and IRMPD spectroscopy, along with computational chemistry, was applied to analyze and evaluate the structure of resin-bound glutathione. Two-dimensional 1H-1H NMR experiments such as COSY, TOCSY, and ROESY were employed to assign and predict the structure of the resin-bound peptide. IRMPD results were used along with calculated protonated structures and spectra to evaluate the conformation of the peptide. The experimental spectrum was compared to the spectra and structures of the protonated species to hypothesize the most favoured structure. Molecular mechanics, molecular dynamics and DFT calculations were implemented to collect structures that best resembled the free and resin-bound glutathione peptide. The results from these methods were compared to determine the structure that is most probable for the glutathione peptide. A semi-folded conformation is the structure the resin-bound GSH most preferred as concluded from the NMR and DFT results. The IRMPD results were analyzed as separate from the resin-bound experiments and suggested protonated GSH had a folded conformation. FK-13 was another peptide synthesized using the solid-phase peptide synthesis technique. The peptide was synthesized using a modified technique different from conventional methodology used in the past. The peptide was also analyzed using COSY, TOCSY, and ROESY to confirm that the synthesis was done correctly and hypothesize a structure. The low substitution of the peptide on the resin gave rise to minimal NOE interactions, but there was some evidence suggesting that the synthesis was successful and the peptide adopted a cyclic conformation. These initial results are useful for future analyses and conformational studies of this resin-bound peptide. Further work needs to be done for both peptides to explore the structures in more detail. The explicit model of solvation should be used to explore the effect of solvent molecules on the conformation of the glutathione peptide as opposed to the implicit model that PCM provides. FK-13 could be synthesized better so that a higher substitution is achieved and better NMR results are obtained. The IRMPD results obtained by the McMahon group can then be compared to the NMR results and computational calculations can be performed to obtain realistic structures of the peptide.