Effects of solute-matrix interaction on monitoring the conformational changes of immobilized proteins by surface plasmon resonance sensor

A real-time and labeling-free surface plasmon resonance (SPR) sensor was used to monitor the conformational changes of immobilized globule proteins (RNase A and lysozyme) in chemical unfolding and refolding. The effects of chemical denaturants on the protein structures were investigated. The methodology in protein conformational study on the solid surface is refined through the theoretic calculations and the conformational information of native/denatured proteins in solution. Additionally, our observation illustrates that the ambient buffer solution is merit to influence the refractive index of immobilized protein films and directly be observed from the SPR resonance angle shifts.     

Interpreting protein structural dynamics from NMR chemical shifts

In this investigation, semiempirical NMR chemical shift prediction methods are used to evaluate the dynamically averaged values of backbone chemical shifts obtained from unbiased molecular dynamics (MD) simulations of proteins. MD-averaged chemical shift predictions generally improve agreement with experimental values when compared to predictions made from static X-ray structures. Improved chemical shift predictions result from population-weighted sampling of multiple conformational states and from sampling smaller fluctuations within conformational basins. Improved chemical shift predictions also result from discrete changes to conformations observed in X-ray structures, which may result from crystal contacts, and are not always reflective of conformational dynamics in solution. Chemical shifts are sensitive reporters of fluctuations in backbone and side chain torsional angles, and averaged (1)H chemical shifts are particularly sensitive reporters of fluctuations in aromatic ring positions and geometries of hydrogen bonds. In addition, poor predictions of MD-averaged chemical shifts can identify spurious conformations and motions observed in MD simulations that may result from force field deficiencies or insufficient sampling and can also suggest subsets of conformational space that are more consistent with experimental data. These results suggest that the analysis of dynamically averaged NMR chemical shifts from MD simulations can serve as a powerful approach for characterizing protein motions in atomistic detail.     

Cu2+-cyclen as probe to identify conformational states in guanine nucleotide binding proteins

(31)P NMR spectroscopy is a suitable method for identifying conformational states in the active site of guanine nucleotide binding proteins detecting the nucleotide placed there. Because there is no labeling necessary, this method is gaining increasing interest. By (31)P NMR spectroscopy two major conformational states, namely state 1(T) and state 2(T), can be detected in active Ras protein characterized by different chemical shifts. Depending on the conformational state Ras shows clearly different physiological properties. Meanwhile analogous conformational equilibria could also be shown for other members of the Ras superfamily. It is often difficult to determine the conformational states of the proteins on the basis of chemical shift alone; therefore, direct detection would be a great advantage. With the use of Cu(2+)-cyclen which selectively interacts only with one of the major conformational states (state 1) one has a probe to distinguish between the two states, because only proteins existing in conformational state 1 interact with the Cu(2+)-cyclen at low millimolar concentrations. The suitability was proven using Ras(wt) and Ras mutants, Ras complexed with GTP, GppNHp, or GTPγS, as well as two further members of the Ras superfamily namely Arf1 and Ran.     

Mapping the potential energy landscape of intrinsically disordered proteins at amino Acid resolution

Intrinsically disordered regions are predicted to exist in a significant fraction of proteins encoded in eukaryotic genomes. The high levels of conformational plasticity of this class of proteins endows them with unique capacities to act in functional modes not achievable by folded proteins, but also places their molecular characterization beyond the reach of classical structural biology. New techniques are therefore required to understand the relationship between primary sequence and biological function in this class of proteins. Although dependences of some NMR parameters such as chemical shifts (CSs) or residual dipolar couplings (RDCs) on structural propensity are known, so that sampling regimes are often inferred from experimental observation, there is currently no framework that allows for a statistical mapping of the available Ramachandran space of each amino acid in terms of conformational propensity. In this study we develop such an approach, combining highly efficient conformational sampling with ensemble selection to map the backbone conformational sampling of IDPs on a residue specific level. By systematically analyzing the ability of NMR data to map the conformational landscape of disordered proteins, we identify combinations of RDCs and CSs that can be used to raise conformational degeneracies inherent to different data types, and apply these approaches to characterize the conformational behavior of two intrinsically disordered proteins, the K18 domain from Tau protein and N(TAIL) from measles virus nucleoprotein. In both cases, we identify the enhanced populations of turn and helical regions in key regions of the proteins, as well as contiguous strands that show clear and enhanced polyproline II sampling.     

Characterization of protein-ligand interactions by high-resolution solid-state NMR spectroscopy

A novel approach for detection of ligand binding to a protein in solid samples is described. Hydrated precipitates of the anti-apoptotic protein Bcl-xL show well-resolved (13)C-(13)C 2D solid-state NMR spectra that allow site-specific assignment of resonances for many residues in uniformly (13)C-enriched samples. Binding of a small peptide or drug-like organic molecule leads to changes in the chemical shift of resonances from multiple residues in the protein that can be monitored to characterize binding. Differential chemical shifts can be used to distinguish between direct protein-ligand contacts and small conformational changes of the protein induced by ligand binding. The agreement with prior solution-state NMR results indicates that the binding pocket in solid and liquid samples is similar for this protein. Advantages of different labeling schemes involving selective (13)C enrichment of methyl groups of Ala, Val, Leu, and Ile (Cdelta1) for characterizing protein-ligand interactions are also discussed. It is demonstrated that high-resolution solid-state NMR spectroscopy on uniformly or extensively (13)C-enriched samples has the potential to screen proteins of moderate size ( approximately 20 kDa) for ligand binding as hydrated solids. The results presented here suggest the possibility of using solid-state NMR to study ligand binding in proteins not amenable to solution NMR.     

Sequence-dependent correction of random coil NMR chemical shifts.

Random coil chemical shifts are commonly used to detect secondary structure elements in proteins in chemical shift index calculations. While this technique is very reliable for folded proteins, application to unfolded proteins reveals significant deviations from measured random coil shifts for certain nuclei. While some of these deviations can be ascribed to residual structure in the unfolded protein, others are clearly caused by local sequence effects. In particular, the amide nitrogen, amide proton, and carbonyl carbon chemical shifts are highly sensitive to the local amino acid sequence. We present a detailed, quantitative analysis of the effect of the 20 naturally occurring amino acids on the random coil shifts of (15)N(H), (1)H(N), and (13)CO resonances of neighboring residues, utilizing complete resonance assignments for a set of five-residue peptides Ac-G-G-X-G-G-NH(2). The work includes a validation of the concepts used to derive sequence-dependent correction factors for random coil chemical shifts, and a comprehensive tabulation of sequence-dependent correction factors that can be applied for amino acids up to two residues from a given position. This new set of correction factors will have important applications to folded proteins as well as to short, unstructured peptides and unfolded proteins.     

Rapid calculation of protein chemical shifts using bond polarization theory and its application to protein structure refinement

Although difficult to analyze, NMR chemical shifts provide detailed information on protein structure. We have adapted the semi-empirical bond polarization theory (BPT) to protein chemical shift calculation and chemical shift driven protein structure refinement. A new parameterization for BPT amide nitrogen chemical shift calculation has been derived from MP2 ab initio calculations and successfully evaluated using crystalline tripeptides. We computed the chemical shifts of the small globular protein ubiquitin, demonstrating that BPT calculations can match the results obtained at the DFT level of theory at very low computational cost. In addition to the calculation of chemical shift tensors, BPT allows the calculation of chemical shift gradients and consequently chemical shift driven geometry optimizations. We applied chemical shift driven protein structure refinement to the conformational analysis of a set of Trypanosoma brucei (the causative agent of African sleeping sickness) tryparedoxin peroxidase Px III structures. We found that the interaction of Px III with its reaction partner Tpx seems to be governed by conformational selection rather than by induced fit.     

蛋白质分子与配体结合过程构象变化的研究

蛋白质分子与配体的作用模式主要有直接的环区结合及铰链式结合两种方式。针对这两种不同的作用方式,我们提出采用不同的策略进行结合过程的构象研究。对于直接的环区结合模式,通过建立环区主链构象库,来实现蛋白质环区与配体的准柔性对接,并以链霉抗生物素蛋白体系为例对构象库建立的可行性进行了验证计算。对铰链结合方式,采用分步对接的方法进行计算,并具体应用于HIV蛋白酶与其小分子配体的结合过程。计算结果表明,这两种处理方法分别能较好地模拟不同类型的蛋白质与配体结合的的构象变化。     

Microcantilever biosensors based on conformational change of proteins

Microcantilevers (MCLs) hold a position as a cost-effective and highly sensitive sensor platform for medical diagnostics, environmental analysis and fast throughput analysis. MCLs are unique in that adsorption of analytes on the microcantilever (MCL) surface changes the surface characteristics of the MCL and results in bending of the MCL. Surface stress due to conformation change of proteins and other polymers has been a recent focus of MCL research. Since conformational changes in proteins can be produced through binding of anylates at specific receptor sites, MCLs that respond to conformational change induced surface stress are promising as transducers of chemical information and are ideal for developing microcantilever-based biosensors. The MCL can also potentially be used to investigate conformational change of proteins induced by non-binding events such as post-translational modification and changes in temperature or pH. This review will provide an overview of MCL biosensors based on conformational change of proteins bound to the MCL surface. The models include conformational change of proteins, proteins on membranes, enzymes, DNA and other polymers.     

Site-specific incorporation of a (19)F-amino acid into proteins as an NMR probe for characterizing protein structure and reactivity

19F NMR is a powerful tool for monitoring protein conformational changes and interactions; however, the inability to site-specifically introduce fluorine labels into proteins of biological interest severely limits its applicability. Using methods for genetically directing incorporation of unnatural amino acids, we have inserted trifluoromethyl-l-phenylalanine (tfm-Phe) into proteins in vivo at TAG nonsense codons with high translational efficiency and fidelity. The binding of substrates, inhibitors, and cofactors, as well as reactions in enzymes, were studied by selective introduction of tfm-Phe and subsequent monitoring of the 19F NMR chemical shifts. Subtle protein conformational changes were detected near the active site and at long distances (25 Angstrom). 19F signal sensitivity and resolution was also sufficient to differentiate protein environments in vivo. Since there has been interest in using 19F-labeled proteins in solid-state membrane protein studies, folding studies, and in vivo studies, this general method for genetically incorporating a 19F-label into proteins of any size in Escherichia coli should have broad application beyond that of monitoring protein conformational changes.     

The use of mid-points or average NMR chemical shifts in stereochemical assignments

A comparison of the mid-points or average chemical shifts of mirror-symmetrical spin patterns in the NMR spectra of structural isomers can be used in a straightforward manner to obtain stereochemical information. This result is anticipated from analysis of substituent contributions to chemical shifts and has been observed in a variety of chemical systems, especially cyclobutane derivatives, which comprise a group of compounds for which appreciable data is available and whose structure assignments have often entailed difficulty and even controversy. The method of mid-point comparison may also be useful for conformational analysis.     

Structure determination of protein-protein complexes using NMR chemical shifts: case of an endonuclease colicin-immunity protein complex

Nuclear magnetic resonance (NMR) spectroscopy provides a range of powerful techniques for determining the structures and the dynamics of proteins. The high-resolution determination of the structures of protein-protein complexes, however, is still a challenging problem for this approach, since it can normally provide only a limited amount of structural information at protein-protein interfaces. We present here the determination using NMR chemical shifts of the structure (PDB code 2K5X) of the cytotoxic endonuclease domain from bacterial toxin colicin (E9) in complex with its cognate immunity protein (Im9). In order to achieve this result, we introduce the CamDock method, which combines a flexible docking procedure with a refinement that exploits the structural information provided by chemical shifts. The results that we report thus indicate that chemical shifts can be used as structural restraints for the determination of the conformations of protein complexes that are difficult to obtain by more standard NMR approaches.     

Molecular simulation to characterize the adsorption behavior of a fibrinogen gamma-chain fragment

Implants invoke inflammatory responses from the body even if they are chemically inert and nontoxic. It has been shown that a crucial precedent event in the inflammatory process is the spontaneous adsorption of fibrinogen (Fg) on implant surfaces, which is typically followed by the presence of phagocytic cells. Interactions between the phagocyte integrin Mac-1 and two short sequences within the fibrinogen gamma chain, gamma190-202 and gamma377-395, may partially explain phagocyte accumulation at implant surfaces. These two sequences are believed to form an integrin binding site that is inaccessible when Fg is in its soluble-state structure but then becomes available for Mac-1 binding following adsorption, presumably due to adsorption-induced conformational changes. The objective of this research was to theoretically investigate this possibility by using molecular dynamics simulations of the gamma-chain fragment of Fg over self-assembled monolayer (SAM) surfaces presenting different types of surface chemistry. The GROMACS software package was used to carry out the molecular simulations in an explicit solvation environment over a 5 ns period of time. The adsorption of the gamma-chain of fibrinogen was simulated on five types of SAM surfaces. The simulations showed that this protein fragment exhibits distinctly different adsorption behavior on the different surface chemistries. Although the trajectory files showed that significant conformational changes did not occur in this protein fragment over the time frame of the simulations, it was predicted that the protein does undergo substantial rotational and translational motions over the surface prior to stabilizing in various preferred orientations. This suggests that the kinetics of surface-induced conformational changes in a protein's structure might be much slower than the kinetics of orientational changes, thus enabling the principles of adsorption thermodynamics to be used to guide adsorbing proteins into defined orientations on surfaces before large conformational changes can occur. This finding may be very important for biomaterial surface design as it suggests that surface chemistry can potentially be used to directly control the orientation of adsorbing proteins in a manner that either presents or hides specific bioactive sites contained within a protein's structure, thereby providing a mechanism to control cellular responses to the adsorbed protein layer.     

Dependence of amino acid side chain 13C shifts on dihedral angle: application to conformational analysis

Chemical shift data from the BiomagResDataBank and conformational data derived from the protein data bank have been correlated in order to explore the conformational dependence of side chain (13)C resonance shifts. Consistent with predictions based on steric compression, upfield shifts for Cgamma resonances of Thr, Val, Ile, Leu, Met, Arg, Lys, Glu, and Gln residues correlate with both the number of heavy atom (nonproton) gamma-substituents and with gauche conformational orientations of gamma-substituents. The (13)C shift/conformation correlations are most apparent for Cgamma carbons but also can be observed at positions further from the backbone. Intraresidue steric conflict leads to a correlation between upfield-shifted side chain (13)C resonances and statistically lower probabilities in surveys of protein side chain conformation. Illustrative applications to the DNA pol lambda lyase domain and to dihydrofolate reductase are discussed. In the latter case, (13)C shift analysis indicates that the conformation of the remote residue V119 on the betaF-betaG loop is correlated with the redox state of the bound pyridine nucleotide cofactor, providing one basis for discrimination between substrate and product. It is anticipated that (13)C shift data for protein sidechains can provide a useful basis for the analysis of conformational changes even in large, deuterated proteins. Additionally, the large dependence of the leucine methyl shift difference, deltaCdelta1-deltaCdelta2, on both chi1 and chi2 is sufficient to allow this parameter to be used as a restraint in structure calculations if stereospecific assignment data are available.     

Predicting chemical shifts with graph neural networks

Inferring molecular structure from Nuclear Magnetic Resonance (NMR) measurements requires an accurate forward model that can predict chemical shifts from 3D structure. Current forward models are limited to specific molecules like proteins and state-of-the-art models are not differentiable. Thus they cannot be used with gradient methods like biased molecular dynamics. Here we use graph neural networks (GNNs) for NMR chemical shift prediction. Our GNN can model chemical shifts accurately and capture important phenomena like hydrogen bonding induced downfield shift between multiple proteins, secondary structure effects, and predict shifts of organic molecules. Previous empirical NMR models of protein NMR have relied on careful feature engineering with domain expertise. These GNNs are trained from data alone with no feature engineering yet are as accurate and can work on arbitrary molecular structures. The models are also efficient, able to compute one million chemical shifts in about 5 seconds. This work enables a new category of NMR models that have multiple interacting types of macromolecules.

This model can predict chemical shifts on proteins and small molecules purely from atom elements and coordinates. It can capture important phenomena like hydrogen bonding induced downfield shift, thus can be used to infer intermolecular interactions.     

13C NMR spectroscopy of core heme carbons as a simple tool to elucidate the coordination state of ferric high-spin heme proteins

Evidence is presented demonstrating that the magnitudes of the 13C chemical shifts originating from heme meso carbons provide a straightforward diagnostic tool to elucidate the coordination state of high-spin heme proteins and enzymes. Pentacoordinate high-spin heme centers exhibit 13C meso shifts centered at approximately 250 ppm, whereas their hexacoordinate counterparts exhibit 13C shifts centered at approximately -80 ppm. The relatively small spectral window (400 to -100 ppm) covering the meso-13C shifts, the relatively narrow lines of these resonances, and the availability of biosynthetic methods to prepare 13C-labeled heme (Rivera, M.; Walker, F. A. Anal. Biochem. 1995, 230, 295-302) make this approach practical. The theoretical basis for the distinct chemical shifts observed for meso carbons from hexacoordinate high-spin hemes relative to their pentacoordinate counterparts are now well understood (Cheng, R.-J.; Chen, P. Y.; Lovell, T.; Liu, T.; Noodleman, L.; Case, D. A. J. Am. Chem. Soc. 2003, 125, 6774-6783), which indicates that the magnitude of the meso-carbon chemical shifts can be used as a simple and reliable diagnostic tool for determining the coordination state of the heme active sites, independent of the nature of the proximal ligand. Proof of the principle for the 13C NMR spectroscopic approach is demonstrated using hexa- and pentacoordinate myoglobin. Subsequently, 13C NMR spectroscopy has been used to unambiguously determine that a recently discovered heme protein from Shigella dysenteriae (ShuT) is pentacoordinate.     

Effects of guanidinium ions on the conformational structure of glucose oxidase studied by electrochemistry, spectroscopy, and theoretical calculations: towards developing a chemical-induced protein conformation assay

Understanding conformation transitions of proteins in the presence of a chemical denaturant is a topic of great interest because the rich information contained in chemical unfolding is of fundamental importance for proteomic and pharmaceutical research. In this work, the conformational structure changes of glucose oxidase (GOx) induced by guanidinium ions (Gdm(+)) were studied in detail by a combination of electrochemical methods, various spectroscopic techniques including ultraviolet-visible (UV-vis) absorption, fluorescence, Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopy, molecular dynamics (MD) simulations, and density functional theory (DFT) calculations with the purpose of revealing the mechanism of chemical unfolding of proteins. The results indicated that GOx underwent substantial conformational changes both at the secondary and tertiary structure levels after interacting with Gdm(+) ions. The interaction of GOx with the chemical denaturant resulted in a disturbance of the structure of the flavin prosthetic group (FAD moiety) that induced the moiety to become less exposed to solvent than that in the native protein molecule. The calculation from quantitative second-derivative infrared and CD spectra showed that Gdm(+) ions induced the conversion of α-helix to β-sheet structures. MD simulations and DFT calculations revealed that Gdm(+) ions could enter the active pocket of the GOx molecule and interact with the FAD group, leading to a significant alteration in the structural characteristics and hydrogen bond networks formed between FAD and the surrounding amino acid residues. These alterations in the conformational structure of GOx resulted in a significant decrease in the catalytic activity of the enzyme to glucose oxidation. The study essentially provides an effective way for investigating the mechanism of chemical denaturant-induced protein unfolding, and this approach can be used for assessing the effect of drug molecules on proteins.     

Carbon-13 NMR and conformational analysis of meso- and dl-α,α′-disubstituted succinic acids

meso-and dl-Diastereomers of a number of α,α′-disubstituted succinic acids have been shown to give different ¹³C NMR chemical shifts. The results can be satisfactorily explained on the basis of their conformational analyses. A discussion of the observed chemical shifts is presented, and the preferred conformation for each of several compounds is predicted on the basis of these chemical shifts.     

Use of 13Calpha chemical shifts in protein structure determination

A physics-based method aimed at determining protein structures by using NOE-derived distances together with observed and computed 13C chemical shifts is proposed. The approach makes use of 13Calpha chemical shifts, computed at the density functional level of theory, to obtain torsional constraints for all backbone and side-chain torsional angles without making a priori use of the occupancy of any region of the Ramachandran map by the amino acid residues. The torsional constraints are not fixed but are changed dynamically in each step of the procedure, following an iterative self-consistent approach intended to identify a set of conformations for which the computed 13Calpha chemical shifts match the experimental ones. A test is carried out on a 76-amino acid, all-alpha-helical protein; namely, the Bacillus subtilis acyl carrier protein. It is shown that, starting from randomly generated conformations, the final protein models are more accurate than an existing NMR-derived structure model of this protein, in terms of both the agreement between predicted and observed 13Calpha chemical shifts and some stereochemical quality indicators, and of similar accuracy as one of the protein models solved at a high level of resolution. The results provide evidence that this methodology can be used not only for structure determination but also for additional protein structure refinement of NMR-derived models deposited in the Protein Data Bank.