Solid-state NMR structural studies of peptides immobilized on gold nanoparticles

In this paper we describe solid-state NMR experiments that provide information on the structures of surface-immobilized peptides. The peptides are covalently bound to alkanethiolates that are self-assembled as monolayers on colloidal gold nanoparticles. The secondary structure of the immobilized peptides was characterized by quantifying the Ramachandran angles phi and psi. These angles were determined in turn from distances between backbone carbonyl 13C spins, measured with the double-quantum filtered dipolar recoupling with a windowless sequence experiment, and by determination of the mutual orientation of chemical shift anisotropy tensors of 13C carbonyl spins on adjacent peptide planes, obtained from the double-quantum cross-polarization magic-angle spinning spectrum. It was found that peptides composed of periodic sequences of leucines and lysines were bound along the length of the peptide sequence and displayed a tight alpha-helical secondary structure on the gold nanoparticles. These results are compared to similar studies of peptides immobilized on hydrophobic surfaces.     

Determination of peptide backbone torsion angles using double-quantum dipolar recoupling solid-state NMR spectroscopy

Several approaches for utilizing dipolar recoupling solid-state NMR (ssNMR) techniques to determine local structure at high resolution in peptides and proteins have been developed. However, many of these techniques measure only one torsion angle or are accurate for only certain classes of secondary structure. Additionally, the efficiency with which these dipolar recoupling experiments suppress the deleterious effects of chemical shift anisotropy (CSA) at high magnetic field strengths varies. Dipolar recoupling with a windowless sequence (DRAWS) has proven to be an effective pulse sequence for exciting double-quantum (DQ) coherences between adjacent carbonyl carbons along the peptide backbone. By allowing this DQ coherence to evolve, it is possible to measure the relative orientations of the CSA tensors and subsequently use this information to determine the Ramachandran torsion angles phi and psi. Here, we explore the accuracies of the assumptions made in interpreting DQ-DRAWS data and demonstrate their fidelity in measuring torsion angles corresponding to a variety of secondary structures irrespective of hydrogen-bonding patterns. It is shown how a simple choice of isotopic labels and experimental conditions allows accurate measurement of backbone secondary structures without any prior knowledge. This approach is considerably more sensitive for determining structure in helices and has comparable accuracy for beta-sheet and extended conformations relative to other methods. We also illustrate the ability of DQ-DRAWS to distinguish between structures in heterogeneous samples.     

Quantification of surface-bound proteins by fluorometric assay: Comparison with quartz crystal microbalance and amido black assay

Protein adsorption is of major and widespread interest, being useful in the fundamental understanding of biological processes at interfaces through to the development of new materials. A number of techniques are commonly used to study protein adhesion, but few are directly quantitative. Here we describe the use of Nano Orange, a fluorometric assay, to quantitatively assess the adsorption of bovine fibrinogen and albumin onto model hydrophilic (OH terminated) and hydrophobic (CH3 terminated) surfaces. Results obtained using this method allowed the calibration of previously unquantifiable data obtained on the same surfaces using quartz crystal microbalance measurements and an amido black protein assay. Both proteins were found to adsorb with higher affinity but with lower saturation levels onto hydrophobic surfaces. All three analytical techniques showed similar trends in binding strength and relative amounts adsorbed over a range of protein concentrations, although the fluorometric analysis was the only method to give absolute quantities of surface-bound protein. The versatility of the fluorometric assay was also probed by analyzing protein adsorption onto porous superhydrophobic and superhydrophilic surfaces. Results obtained using the assay in conjunction with these surfaces were surface chemistry dependent. Imbibition of water into the superhydrophilic coatings provided greater surface area for protein adsorption, although the protein surface density was less than that found on a comparable flat hydrophilic surface. Superhydrophobic surfaces prevented protein solution penetration. This paper demonstrates the potential of a fluorometric assay to be used as an external calibration for other techniques following protein adsorption processes or as a supplemental method to study protein adsorption. Differences in protein adsorption onto hydrophilic vs superhydrophilic and hydrophobic vs superhydrophobic surfaces are highlighted.     

Solid-state NMR spectroscopic methods in chemistry

Over the last decades, NMR spectroscopy has grown into an indispensable tool for chemical analysis, structure determination, and the study of dynamics in organic, inorganic, and biological systems. It is commonly used for a wide range of applications from the characterization of synthetic products to the study of molecular structures of systems such as catalysts, polymers, and proteins. Although most NMR experiments are performed on liquid-state samples, solid-state NMR is rapidly emerging as a powerful method for the study of solid samples and materials. This Review outlines some of the developments of solid-state NMR spectroscopy, including techniques such as cross-polarization, magic-angle spinning, multiple-pulse sequences, homo- and heteronuclear decoupling and recoupling techniques, multiple-quantum spectroscopy, and dynamic angle spinning, as well as their applications to structure determination. Modern solid-state NMR spectroscopic techniques not only produce spectra with a resolution close to that of liquid-state spectra, but also capitalize on anisotropic interactions, which are often unavailable for liquid samples. With this background, the future of solid-state NMR spectroscopy in chemistry appears to be promising, indeed.     

The initial steps in the hydration of unsolvated peptides: water molecule adsorption on alanine-based helices and globules

Equilibrium constants for the adsorption of the first water molecule onto a variety of unsolvated alanine-based peptides have been measured and Delta H degrees and DeltaS degrees have been determined. The studies were designed to examine the effects of conformation, charge, and composition on the propensity for peptides to bind water. In general, water adsorption occurs significantly more readily on the globular peptides than on helical ones: several of the singly charged helical peptides were not observed to adsorb a water molecule even at -50 degrees C. These results place a limit on the free energy change for interaction between a water molecule and the helical peptide group. Molecular dynamics simulations reproduce most of the main features of the results. The ability to establish a network of hydrogen bonds to several different hydrogen-bonding partners emerges as a critical factor for strong binding of the water molecule. Whether the charge site is involved in water adsorption depends on how well it is shielded. Peptides containing a protonated histidine bind water much more strongly that those containing a protonated lysine because the delocalized charge on histidine is difficult to shield. The entropy change for adsorption of the first water molecule is correlated with the enthalpy change.     

Benzoxazine oligomers: evidence for a helical structure from solid-state NMR spectroscopy and DFT-based dynamics and chemical shift calculations

A combination of molecular modeling, DFT calculations, and advanced solid-state NMR experiments is used to elucidate the supramolecular structure of a series of benzoxazine oligomers. Intramolecular hydrogen bonds are characterized and identified as the driving forces for ring-shape and helical conformations of trimeric and tetrameric units. In fast MAS (1)H NMR spectra, the resonances of the protons forming the hydrogen bonds can be assigned and used for validating and refining the structure by means of DFT-based geometry optimizations and (1)H chemical-shift calculations. Also supporting these proposed structures are homonuclear (1)H[bond](1)H double-quantum NMR spectra, which identify the local proton-proton proximities in each material. Additionally, quantitative (15)N[bond](1)H distance measurements obtained by analysis of dipolar spinning sideband patterns confirm the optimized geometry of the tetramer. These results clearly support the predicted helical geometry of the benzoxazine polymer. This geometry, in which the N...H...O and O...H...O hydrogen bonds are protected on the inside of the helix, can account for many of the exemplary chemical properties of the polybenzoxazine materials. The combination of advanced experimental solid-state NMR spectroscopy with computational geometry optimizations, total energy, and NMR spectra calculations is a powerful tool for structural analysis. Its results provide significantly more confidence than the individual measurements or calculations alone, in particular, because the microscopic structure of many disordered systems cannot be elucidated by means of conventional methods due to lack of long-range order.     

Segmental dynamics of PEO/LiClO4 complex crystals and their influence on the Li+-ion transportation in crystal lattices: A 13C solid-state NMR approach

The structure and chain dynamics of polyethylene oxide (PEO)/LiClO(4) complex crystals were studied by employing state-of-the-art solid-state (13)C NMR techniques. Remarkable helical jump motions of the PEO segments in the complex crystals at ambient temperatures were clearly demonstrated. The jump motions are believed to induce the transportation of the coordinated Li(+) ions along the crystallographic c axis, providing a novel mechanism of ionic conductivity of the complex crystals. In addition, this work also shows that solid-state high-resolution (13)C NMR spectroscopy can be a powerful and general tool for elucidating phase structures, dynamics, and subsequently the conduction mechanism of crystalline polymer electrolytes.     

Toward an understanding of the formation of vanadia-titania catalysts

Structures of hydrated vanadia species on the TiO2-anatase surfaces were investigated using the semiempirical molecular orbital method MSINDO. The (101), (001), and (100) surfaces of anatase were considered. They were modeled by appropriate two-dimensional cyclic clusters of TiO2. Monomeric and dimeric hydrated vanadia species on the anatase surfaces were simulated by adsorbing VO4H3 and V2O7H4 molecules, respectively. Different adsorption structures were considered, and their stabilities at 300 and 600 K were tested by constant-temperature Born-Oppenheimer molecular dynamics simulations in the framework of MSINDO. Structural features of the vanadia-titania catalysts found in extended X-ray absorption fine structure, secondary ion mass spectrometry, IR, Raman, and NMR spectroscopy and conductivity experiments can be explained by the present calculations.     

Sequence, structure, and function of peptide self-assembled monolayers

Cysteine is commonly used to attach peptides onto gold surfaces. Here we show that the inclusion of an additional linker with a length of four residues (-PPPPC) and a rigid, hydrophobic nature is a better choice for forming peptide self-assembled monolayers (SAMs) with a well-ordered structure and high surface density. We compared the structure and function of the nonfouling peptide EKEKEKE-PPPPC-Am with EKEKEKE-C-Am. Circular dichroism, attenuated total internal reflection Fourier transform IR spectroscopy, and molecular dynamics results showed that EKEKEKE-PPPPC-Am forms a secondary structure while EKEKEKE-C-Am has a random structure. Surface plasmon resonance sensor results showed that protein adsorption on EKEKEKE-PPPPC-Am/gold is very low with small variation while protein adsorption on EKEKEKE-C-Am/gold is high with large variation. X-ray photoelectron spectroscopy results showed that both peptides have strong gold-thiol binding with the gold surface, indicating that their difference in protein adsorption is due to their assembled structures. Further experimental and simulation studies were performed to show that -PPPPC is a better linker than -PC, -PPC, and -PPPC. Finally, we extended EKEKEKE-PPPPC-Am with the cell-binding sequence RGD and demonstrated control over specific versus nonspecific cell adhesion without using poly(ethylene glycol). Adding a functional peptide to the nonfouling EK sequence avoids complex chemistries that are used for its connection to synthetic materials.     

Assigning powders to crystal structures by high-resolution (1)H-(1)H double quantum and (1)H-(13)C J-INEPT solid-state NMR spectroscopy and first principles computation. A case study of penicillin G

We show how powder samples at natural isotopic abundance can be assigned to crystal structures by using high-resolution proton and carbon-13 solid-state NMR spectra in combination with first principles calculations. Homonuclear proton double-quantum spectra in combination with through-bond proton-carbon HSQC spectra are used to assign the NMR spectra. We then show that the proton chemical shifts can be included in the process of assigning the spectra to a crystal structure using first principles calculations. The method is demonstrated on the K salt of penicillin G.     

Understanding CO2 adsorption mechanisms in porous adsorbents: A solid-state NMR survey

Reaching a historic high of 36.3 gigatonnes in 2021, global CO₂ emissions from fossil fuel combustion continue to increase at an alarming rate. CO₂ removal technologies are part of the solution to tackle this crucial environmental challenge. Thus, the development of porous materials for storage and capture of gas molecules (in particular, carbon capture and storage) has attracted great interest both in academic and industrial communities due to its potential in mitigating atmospheric CO₂ concentrations.Atomic-scale studies in porous materials are essential to stimulate progress in the design of better CO₂-adsorbents by elucidating gas-sorption surface mechanisms.Spectroscopic techniques have the potential to shed light on the structural details of distinct materials' surfaces, including the type of chemical species formed upon CO₂ adsorption. Herein, we review the last 5 years of scientific developments wherein solid-state NMR and computational studies have been explored to investigate at the atomic level the structure and molecular dynamics of CO₂ species formed at porous surfaces.     

The interaction of lipid modified pseudopeptides with lipid membranes

We have studied the structure of two lipopeptides based on the simple dipeptide building block L-Phe-D-Oxd. These peptides have been reported previously to form fiber-like materials. The lipopeptides synthesized here had the structures C(n)(2)H((2n+1))CO-L-Phe-D-Oxd-OBn or C(n)(2)H((2n+1))CO-D-Phe-L-Oxd-OBn with n = 5 or 11. Addition of the N-terminal lipid modification did not cause a major disturbance of the structures these molecules form. The lipid modifications themselves showed highly rigid structures as inferred from solid-state (2)H NMR. The peptide backbone showed (13)C NMR chemical shifts in agreement with β-sheet secondary structure. Addition of a lipid modification to the N-terminus is a common motif in biology to attach proteins to the membrane. Therefore, we also investigated the lipopeptides in the presence of synthetic POPC bilayers. Two different molecular species were detected under these circumstances: (i) lipopeptide monomers that showed chain order parameters similar to those of the host membrane, (ii) lipopeptide aggregates that exhibited very similar structures and dynamics as the crystalline aggregates. Overall, the lipopeptides showed a well defined and rigid secondary structure that is in agreement with fibrillar aggregates previously detected for those peptides without the lipid modification.     

Adsorption behavior of statherin and a statherin peptide onto hydroxyapatite and silica surfaces by in situ ellipsometry

The salivary protein statherin is known to adsorb selectively onto hydroxyapatite (HA), which constitutes the main mineral of the tooth enamel. This adsorption is believed to be crucial for its function as an inhibitor of primary (spontaneous) and secondary (crystal growth) precipitation of calcium phosphate salts present in saliva. A fragment corresponding to the first 21 N-terminus amino acids of statherin (StN21) was previously found to reduce the rate of demineralization of HA. Therefore, the interfacial properties of this peptide and statherin onto silica, hydrophobized silica and HA discs was studied by in situ ellipsometry. Their reversibility induced by dilution and elutability induced by buffer and sodium dodecyl sulfate (SDS) was also determined. The results revealed that statherin adsorbed at a greater extent onto the HA as compared to StN21, suggesting that the hydrogen bonding between the uncharged polar residues at the C-terminal region of statherin and HA contributes to its adsorption. However, on both silica surfaces the peptide adsorption appeared to proceed in a similar way. Onto the hydrophobized silica the adsorption of both peptides was suggested to occur either via multilayer formation or adsorption of aggregates from solution, while onto the hydrophilic silica adsorption of peptide aggregates from solution was the suggested mechanism. Further, both peptides were observed to be strongly adsorbed onto HA, even after SDS treatment, in comparison to the layers adsorbed onto hydrophobized silica. Both peptide layers were found to be weakly adsorbed onto the hydrophilic silica surface as they were totally removed by buffer dilution.     

Study of the interaction of human defensins with cell membrane models: relationships between structure and biological activity

The HNP-1, HNP-2, and HNP-3 defensins are human antimicrobial peptides produced in response to microbial invasion. Their properties are distinct, with a more potent action for HNP-3. In this study, the relationship between their structural dissimilarities and their different microbial actions was evaluated by molecular dynamics simulation. Structural determinants related to their intra- and intermolecular interactions were defined for each HNP using a simplified membrane model consisting of a water/n-hexane interface. The hydrophobic portion of the HNPs promotes their diffusion to the interface with a concomitant, slight change in the structure induced by the intermolecular electrostatic interactions between the HPN molecules and the interface. As a consequence, different orientations are probably adopted by the HNPs at the interface, which may explain their different actions. The interaction of HNP-1 and HNP-2 with the surfaces was also studied using Langmuir monolayers as a biomimetic system. It was found that peptides adsorb rapidly at n-hexane/water interfaces as well as at phospholipid Langmuir monolayers but not at the air/liquid interface. This reveals that the presence of an organic phase is required for the exposure of the hydrophobic groups of the peptides. In addition, adsorption kinetics and surface pressure-area isotherms for Langmuir monolayers suggested that the lipid-peptide interaction is strongly influenced by the monolayer electrical charge and packing, depending also on the HPN structure. This study supports a model in which defensins, acting in a dimeric form, are able to disrupt membranes. The model also shows that the individual structures of the HNPs are responsible for their different actions on microbes.     

Structural and dynamical properties of guest molecules confined in mesoporous silica materials revealed by NMR

In the last fifteen years several novel porous silica materials, which are periodically structured on the mesoscopic length scale, have been synthesized. They are of broad interest for fundamental studies of surface-substrate interactions, for studies of the dynamics of guest molecules in confinement and for studies of the effect of confinement on the structural and thermophysical properties of fluids. Examples of such confinement effects include the change of the freezing and melting points or glass transitions of the confined liquids. These effects are studied by combinations of several NMR techniques, such as (15)N- and (2)H-solid-state NMR line shape analysis, MAS NMR and NMR diffusometry with physico-chemical characterization techniques such as nitrogen adsorption and small angle diffraction of neutrons or X-rays. This combination does not require crystalline samples or special clean and well defined surfaces such as conventional surface science techniques, but can work with typical ill-defined real world systems. The review discusses, after a short introduction, the salient features of these materials and the applied NMR experiments to give the reader a basic knowledge of the systems and the experiments. The rest of the review then focuses on the structural and dynamical properties of guest molecules confined in the mesoporous silica. It is shown that the confinement into the pores leads to fascinating new features of the guests, which are often not known for their bulk phases. These features depend strongly on the interplay of the their interactions with the silica surface and their mutual interactions.     

Magic angle spinning and oriented sample solid-state NMR structural restraints combine for influenza a M2 protein functional insights

As a small tetrameric helical membrane protein, the M2 proton channel structure is highly sensitive to its environment. As a result, structural data from a lipid bilayer environment have proven to be essential for describing the conductance mechanism. While oriented sample solid-state NMR has provided a high-resolution backbone structure in lipid bilayers, quaternary packing of the helices and many of the side-chain conformations have been poorly restrained. Furthermore, the quaternary structural stability has remained a mystery. Here, the isotropic chemical shift data and interhelical cross peaks from magic angle spinning solid-state NMR of a liposomal preparation strongly support the quaternary structure of the transmembrane helical bundle as a dimer-of-dimers structure. The data also explain how the tetrameric stability is enhanced once two charges are absorbed by the His37 tetrad prior to activation of this proton channel. The combination of these two solid-state NMR techniques appears to be a powerful approach for characterizing helical membrane protein structure.     

The structure of poly(carbonsuboxide) on the atomic scale: a solid-state NMR study

In this contribution we present a study of the structure of amorphous poly(carbonsuboxide) (C3O2)x by 13C solid-state NMR spectroscopy supported by infrared spectroscopy and chemical analysis. Poly(carbonsuboxide) was obtained by polymerization of carbonsuboxide C3O2, which in turn was synthesized from malonic acid bis(trimethylsilylester). Two different 13C labeling schemes were applied to probe inter- and intramonomeric bonds in the polymer by dipolar solid-state NMR methods and also to allow quantitative 13C MAS NMR spectra. Four types of carbon environments can be distinguished in the NMR spectra. Double-quantum and triple-quantum 2D correlation experiments were used to assign the observed peaks using the through-space and through-bond dipolar coupling. In order to obtain distance constraints for the intermonomeric bonds, double-quantum constant-time experiments were performed. In these experiments an additional filter step was applied to suppress contributions from not directly bonded 13C,13C spin pairs. The 13C NMR intensities, chemical shifts, connectivities and distances gave constraints for both the polymerization mechanism and the short-range order of the polymer. The experimental results were complemented by bond lengths predicted by density functional theory methods for several previously suggested models. Based on the presented evidence we can unambiguously exclude models based on gamma-pyronic units and support models based on alpha-pyronic units. The possibility of planar ladder- and bracelet-like alpha-pyronic structures is discussed.     

Theoretical investigations of oxygen-17 NMR chemical shifts to discriminate among helical forms

17O, 15N, 13C, and 1H NMR chemical shieldings are calculated using density functional theory to differentiate among the three primarily helical forms, 310, alpha, and pi in polyalanine peptides under periodic boundary conditions. This study suggests 17O as the best observable, as it has been demonstrated to be sensitive to hydrogen bonding and highly affected by small changes in the polypeptide in helix conformations. This theoretical study seeks to characterize the subtle conformational differences of helical structures by NMR chemical shift observables which may lead to important questions in experimental structure determination on the basis of using chemical shifts to identify protein secondary structures.     

Construction by molecular dynamics modeling and simulations of the porous structures formed by dextran polymer chains attached on the surface of the pores of a base matrix: characterization of porous structures

Significant increases in the separation of bioactive molecules by using ion-exchange chromatography are realized by utilizing porous adsorbent particles in which the affinity group/ligand is linked to the base matrix of the porous particle via a polymeric extender. To study and understand the behavior of such systems, the M3B model is modified and used in molecular dynamics (MD) simulation studies to construct porous dextran layers on the surface of a base matrix, where the dextran polymer chains and the surface are covered by water. Two different porous polymer layers having 25 and 40 monomers per main polymer chain of dextran, respectively, are constructed, and their three-dimensional (3D) porous structures are characterized with respect to porosity, pore size distribution, and number of conducting pathways along the direction of net transport. It is found that the more desirable practical implications with respect to structural properties exhibited by the porous polymer layer having 40 monomers per main polymer chain, are mainly due to the higher flexibility of the polymer chains of this system, especially in the upper region of the porous structure. The characterization and analysis of the porous structures have suggested a useful definition for the physical meaning and implications of the pore connectivity of a real porous medium that is significantly different than the artificial physical meaning associated with the pore connectivity parameter employed in pore network models and whose physical limitations are discussed; furthermore, the methodology developed for the characterization of the three-dimensional structures of real porous media could be used to analyze the experimental data obtained from high-resolution noninvasive three-dimensional methods like high-resolution optical microscopy. The MD modeling and simulations methodology presented here could be used, considering that the type and size of affinity group/ligand as well as the size of the biomolecule to be adsorbed onto the affinity group/ligand are known, to construct different porous dextran layers by varying the length of the polymeric chain of dextran, the number of attachment points to the base matrix, the degree of side branching, and the number of main polymeric chains immobilized per unit surface area of base matrix. After the characterization of the porous structures of the different porous dextran layers is performed, then only a few promising structures would be selected for studying the immobilization of adsorption sites on the pore surfaces and the subsequent adsorption of the bioactive molecules onto the immobilized affinity groups/ligands.