Monitor Protein Structure Research Articles

Isotope Reverse-Labeled Infrared Spectroscopy as a Probe of In-Cell Protein Structure.

While recent years have seen great progress in determining the three-dimensional structure of isolated proteins, monitoring protein structure inside live cells remains extremely difficult. Here, we examine the utility of Fourier transform infrared (FTIR) spectroscopy as a probe of protein structure in live bacterial cells. Selective isotope enrichment is used both to distinguish recombinantly expressed NuG2b protein from the cellular background and to examine the conformation of specific residues in the protein. To maximize labeling flexibility and to improve spectral resolution between label and main-band peaks, we carry out isotope-labeling experiments in "reverse-labeling" mode: cells are initially grown in 13C-enriched media, with specific 12C-labeled amino acids added when protein expression is induced. 1 Because FTIR measurements require only around 20 μL of sample and each measurement takes only a few minutes to complete, isotope-labeling costs are minimal, allowing us to label multiple different residues in parallel in simultaneously grown cultures. For the stable NuG2b protein, isotope difference spectra from live bacterial cultures are nearly identical to spectra from isolated proteins, confirming that the structure of the protein is unperturbed by the cellular environment. By combining such measurements with site-directed mutagenesis, we further demonstrate that the local conformation of individual amino acids can be monitored, allowing us to determine, for example, whether a specific site in the protein contributes to α-helix or β-sheet structures.

Efficient Analysis of Proteome-Wide FPOP Data by FragPipe.

Monitoring protein structure before and after environmental alterations (e.g., different cell states) can give insights into the role and function of proteins. Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry (MS) allows for monitoring of structural rearrangements by exposing proteins to OH radicals that oxidize solvent-accessible residues, indicating protein regions undergoing movement. Some of the benefits of FPOP include high throughput and a lack of scrambling due to label irreversibility. However, the challenges of processing FPOP data have thus far limited its proteome-scale uses. Here, we present a computational workflow for fast and sensitive analysis of FPOP data sets. Our workflow, implemented as part of the FragPipe computational platform, combines the speed of the MSFragger search with a unique hybrid search method to restrict the large search space of FPOP modifications. Together, these features enable more than 10-fold faster FPOP searches that identify 150% more modified peptide spectra than previous methods. We hope this new workflow will increase the accessibility of FPOP to enable more protein structure and function relationships to be explored.

NIRS to assess chemical composition of sheep and goat cheese

NIRS to assess chemical composition of sheep and goat cheese

Circular-Dichroism and Synchrotron-Radiation Circular-Dichroism Spectroscopy as Tools to Monitor Protein Structure in a Lipid Environment.

Circular-dichroism (CD) spectroscopy is a powerful tool for the secondary-structure analysis of proteins. The structural information obtained by CD does not have atomic-level resolution (unlike X-ray crystallography and NMR spectroscopy), but it has the great advantage of being applicable to both nonnative and native proteins in a wide range of solution conditions containing lipids and detergents. The development of synchrotron-radiation CD (SRCD) instruments has greatly expanded the utility of this method by extending the spectra to the vacuum-ultraviolet region below 190nm and producing information that is unobtainable by conventional CD instruments. Combining SRCD data with bioinformatics provides new insight into the conformational changes of proteins in a membrane environment.

POPPeT: a New Method to Predict the Protection Factor of Backbone Amide Hydrogens

Hydrogen exchange (HX) has become an important tool to monitor protein structure and dynamics. The interpretation of HX data with respect to protein structure requires understanding of the factors that influence exchange. Simulated protein structures can be validated by comparing experimental deuteration profiles with the profiles derived from the modeled protein structure. To do this, we propose here a new method, POPPeT, for protection factor prediction based on protein motions that enable HX. By comparing POPPeT with two existing methods, the phenomenological approximation and COREX, we show enhanced predictability measured at both protection factor and deuteration level. This method can be subsequently used by modeling strategies for protein structure prediction.Graphical ᅟ

Online Hydrogen-Deuterium Exchange Traveling Wave Ion Mobility Mass Spectrometry (HDX-IM-MS): a Systematic Evaluation

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an important tool for measuring and monitoring protein structure. A bottom-up approach to HDX-MS provides peptide level deuterium uptake values and a more refined localization of deuterium incorporation compared with global HDX-MS measurements. The degree of localization provided by HDX-MS is proportional to the number of peptides that can be identified and monitored across an exchange experiment. Ion mobility spectrometry (IMS) has been shown to improve MS-based peptide analysis of biological samples through increased separation capacity. The integration of IMS within HDX-MS workflows has been commercialized but presently its adoption has not been widespread. The potential benefits of IMS, therefore, have not yet been fully explored. We herein describe a comprehensive evaluation of traveling wave ion mobility integrated within an online-HDX-MS system and present the first reported example of UDMSE acquisition for HDX analysis. Instrument settings required for optimal peptide identifications are described and the effects of detector saturation due to peak compression are discussed. A model system is utilized to confirm the comparability of HDX-IM-MS and HDX-MS uptake values prior to an evaluation of the benefits of IMS at increasing sample complexity. Interestingly, MS and IM-MS acquisitions were found to identify distinct populations of peptides that were unique to the respective methods, a property that can be utilized to increase the spatial resolution of HDX-MS experiments by >60%.Graphical ᅟ

Monitoring Protein Structure on the Surface of Gold Nanoparticles using NMR Spectroscopy

Because of their unique spectroscopic properties and biocompatibility, protein-functionalized gold nanoparticles (AuNPs) have many potential diagnostic and therapeutic applications. Surface-bound proteins can be used both as molecular sensors and drug delivery vectors, and the plasmonic properties of gold enable the detection of very small changes in surface chemistry. Unfortunately, the design of general-purpose, functionalized AuNPs is severely complicated by our limited understanding of protein structure on nanoparticle surfaces. Some enzymes remain active on AuNPs while others are inactive, and it is currently impossible to predict which behavior will be observed. To address this problem, we have recently developed several new NMR-based approaches for monitoring protein structure on AuNP surfaces. We find that the adsorption capacity of 15 nm AuNPs can be predicted using the native structure, suggesting that proteins remain globular on the AuNP surface. Additionally, we demonstrate that proteins can be displaced from AuNPs by organothiols, supporting a case for reversible binding. Finally, we have used hydrogen/deuterium exchange (HDX) to monitor structural perturbations of two model proteins, GB3 and Ubiquitin, when bound to AuNPs. We find no significant changes in slow HDX rates (5-300 min), suggesting that AuNP-induced structural changes are small for these two proteins. Together, these results support a model where most of a protein's native contacts are preserved upon adsorption, although larger changes may occur over long timescales.

Using hydrogen-deuterium exchange to monitor protein structure in the presence of gold nanoparticles.

The potential applications of protein-functionalized gold nanoparticles (AuNPs) have motivated many studies characterizing protein-AuNP interactions. However, the lack of detailed structural information has hindered our ability to understand the mechanism of protein adsorption on AuNPs. In order to determine the structural perturbations that occur during adsorption, hydrogen/deuterium exchange (HDX) of amide protons was measured for two proteins by NMR. Specifically, we measured both slow (5-300 min) and fast (10-500 ms) H/D exchange rates for GB3 and ubiquitin, two well-characterized proteins. Overall, amide exchange rates are very similar in the presence and absence of AuNPs, supporting a model where the adsorbed protein remains largely folded on the AuNP surface. Small differences in exchange rates are observed for several loop residues, suggesting that the secondary structure remains relatively rigid while loops and surface residues can experience perturbations upon binding. Strikingly, several of these residues are close to lysines, which supports a model where positive surface residues may interact favorably with AuNP-bound citrate. Because these proteins appear to remain folded on AuNP surfaces, these studies suggest that it may be possible to engineer functional AuNP-based nanoconjugates without the use of chemical linkers.

Deciphering Protein Stability in Cells

Deciphering Protein Stability in Cells

Azido Homoalanine is a Useful Infrared Probe for Monitoring Local Electrostatistics and Side-Chain Solvation in Proteins

The use of IR probes to monitor protein structure, deduce local electric field, and investigate the mechanism of enzyme catalysis and protein folding has attracted increasing attention. Here, the azidohomoalanine (Aha) is considered as a useful IR probe. The intricate details of the distinct effects of backbone peptide bonds and H-bonded water molecules on the azido stretch mode of the IR probe Aha were revealed by carrying out QM/MM MD simulations of two variants of the protein NTL9, NTL9-Met1Aha and NTL9-Ile4Aha and comparing the resulting simulated IR spectra with experiments.

Structural characteristics of the hydrophobic patch of azurin and its interaction with p53: a site-directed spin labeling study

Site-directed spin labeling (SDSL) is a powerful tool for monitoring protein structure, dynamics and conformational changes. In this study, the domain-specific properties of azurin and its interaction with p53 were studied using this technique. Mutations of six residues, that are located in the hydrophobic patch of azurin, were prepared and spin labeled. Spectra of the six azurin mutants in solution showed that spin labeled residues 45 and 63 are in a very restricted environment, residues 59 and 65 are in a spacious environment and have free movement, and residues 49 and 51 are located in a relatively closed pocket. Polarity experiments confirmed these results. The changes observed in the spectra of spin labeled azurin upon interaction with p53 indicate that the hydrophobic patch is involved in this interaction. Our results provide valuable insight into the topographic structure of the hydrophobic domain of azurin, as well as direct evidence of its interaction with p53 in solution via the hydrophobic patch. Cytotoxicity studies of azurin mutants showed that residues along the hydrophobic patch are important for its cytotoxicity.

The effect of the cosolvent trifluoroethanol on a tryptophan side chain orientation in the hydrophobic core of troponin C

The unique biophysical properties of tryptophan residues have been exploited for decades to monitor protein structure and dynamics using a variety of spectroscopic techniques, such as fluorescence and nuclear magnetic resonance (NMR). We recently designed a tryptophan mutant in the regulatory N-domain of cardiac troponin C (F77W-cNTnC) to study the domain orientation of troponin C in muscle fibers using solid-state NMR. In our previous study, we determined the NMR structure of calcium-saturated mutant F77W-V82A-cNTnC in the presence of 19% 2,2,2-trifluoroethanol (TFE). TFE is a widely used cosolvent in the biophysical characterization of the solution structures of peptides and proteins. It is generally assumed that the structures are unchanged in the presence of cosolvents at relatively low concentrations, and this has been verified for TFE at the level of the overall secondary and tertiary structure for several calcium regulatory proteins. Here, we present the NMR solution structure of the calcium saturated F77W-cNTnC in presence of its biological binding partner troponin I peptide (cTnI(144-163)) and in the absence of TFE. We have also characterized a panel of six F77W-cNTnC structures in the presence and absence TFE, cTnI(144-163), and the extra mutation V82A, and used (19)F NMR to characterize the effect of TFE on the F77(5fW) analog. Our results show that although TFE did not perturb the overall protein structure, TFE did induce a change in the orientation of the indole ring of the buried tryptophan side chain from the anticipated position based upon homology with other proteins, highlighting the potential dangers of the use of cosolvents.

Raman Spectroscopy for Monitoring Protein Structure in Muscle Food Systems

Raman spectroscopy offers structural information about complex solid systems such as muscle food proteins. This spectroscopic technique is a powerful and a non-invasive method for the study of protein changes in secondary structure, mainly quantified, analysing the amide I (1650–1680 cm− 1) and amide III (1200–1300 cm− 1) regions and C-C stretching band (940 cm− 1), as well as modifications in protein local environments (tryptophan residues, tyrosil doublet, aliphatic aminoacids bands) of muscle food systems. Raman spectroscopy has been used to determine structural changes in isolated myofibrillar and connective tissue proteins by the addition of different compounds and by the effect of the conservation process such as freezing and frozen storage. It has been also shown that Raman spectroscopy is particularly useful for monitoring in situ protein structural changes in muscle food during frozen storage. Besides, the possibilities of using protein structural changes of intact muscle to predict the protein functional properties and the sensory attributes of muscle foods have been also investigated. In addition, the application of Raman spectroscopy to study changes in the protein structure during the elaboration of muscle food products has been demonstrated.

In this issue

AbstractDiseases of protein aggregationWang et al.: Biotechnol. J. 2008, 3, 165–192Protein aggregation is a ubiquitous phenomenon frequently encountered in biochemical research and the biopharmaceutical industry. It is generally regarded to be associated with partially folded intermediate species that are susceptible to self‐association due to the exposure of a hydrophobic core. Evidence supports the concept that the formation of aggregates in vitro is a generic property of proteins. In human etiology, more than 20 different devastating human diseases have been reported to be associated with protein aggregation in vivo. In this review, researchers from Taiwan and Japan summarize the properties, characteristics and causes of protein aggregates. They briefly discuss two examples of protein aggregation diseases, Alzheimer's disease and cataract. Investigation into drug penetration, efficacy, and side effects will certainly aid in developing successful pharmacological agents for these diseases.Protein recovery from inclusion bodiesDoglia et al.: Biotechnol. J. 2008, 3, 193–201As bacterially overexpressed polypeptides mostly undergo aggregation in inclusion bodies, they have to be recovered by solubilization and refolding procedures. However, recent studies have shown that proteins embedded in inclusion bodies may retain residual structure and biological function and question the former axiom that solubility and activity are necessarily coupled. This allows for a switch in the goals from obtaining soluble products to controlling the conformational quality of aggregated proteins. Central to this approach is the availability of analytical methods to monitor protein structure within inclusion bodies. Researchers from Italy here describe the use of Fourier transform infrared spectroscopy for the structural analysis of inclusion bodies both purified from cells and in vivo. They show kinetics of aggregation and structure of aggregates as a function of expression levels, temperature and co‐expression of chaperones.LC‐MS for protein biosysnthesis analysisJani et al.: Biotechnol. J. 2008, 3, 202–208The optimization of the biosynthetic pathways is highly attractive for the large‐scale preparation of macrotetrolides, because overall yields in the chemical synthesis of compounds like nonactin have been very low. A key success factor determining the outcome of such optimizations is the adequate process analysis for the envisioned product. The analytical methods for process control so far involved spectrophotometric and chromatographic measurements. As shown by the group of Roland Wohlgemuth (Sigma‐Aldrich, Switzerland), LC‐MS offers a modern approach to obtain more detailed data than the spectrophotometric and chromatographic measurements used in the past. LC‐MS combines the advantages of a chromatographic separation with mass detection by MS. Low detection limits and qualified analysis of molecular structures brought this application a new and growing popularity in the field of biotechnology. In this work, a fast and versatile analytical LC‐MS method has been set up. It is used for the in‐process analysis of macrotetrolides during fermentation and allows rapid large‐scale bioprocess development.

Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies

The solubility of recombinant proteins produced in bacterial cells is considered a key issue in biotechnology as most overexpressed polypeptides undergo aggregation in inclusion bodies, from which they have to be recovered by solubilization and refolding procedures. Physiological and molecular strategies have been implemented to revert or at least to control aggregation but they often meet only partial success and have to be optimized case by case. Recent studies have shown that proteins embedded in inclusion bodies may retain residual structure and biological function and question the former axiom that solubility and activity are necessarily coupled. This allows for a switch in the goals from obtaining soluble products to controlling the conformational quality of aggregated proteins. Central to this approach is the availability of analytical methods to monitor protein structure within inclusion bodies. We describe here the use of Fourier transform infrared spectroscopy for the structural analysis of inclusion bodies both purified from cells and in vivo. Examples are reported concerning the study of kinetics of aggregation and structure of aggregates as a function of expression levels, temperature and co-expression of chaperones.

Use of infrared spectroscopy to monitor protein structure and stability

One of the most versatile methods for monitoring the structure of proteins, either in solution or in the solid state, is Fourier transform infrared spectroscopy. Also known as mid-range infrared, which covers the frequency range from 4000 to 400 cm-1, this wavelength region includes bands that arise from three conformationally sensitive vibrations within the peptide backbone (amide I, II and III). Of these vibrations, amide I is the most widely used and can provide information on secondary structure composition and structural stability. One of the advantages of infrared spectroscopy is that it can be used with proteins that are either in solution or in the solid state. The use of infrared to monitor protein structure and stability is summarized herein. In addition, specialized infrared methods are presented, such as techniques for the study of membrane proteins and oriented samples. In addition, there is a growing body of literature on the use of infrared to follow reaction kinetics and ligand binding in proteins, as well as a number of infrared studies on protein dynamics. Finally, the potential for using near-infrared spectroscopy to study protein structure is introduced.

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems.

The recombinant heat shock protein (18 kDa-hsp) from Mycobacterium leprae was studied as a T-epitope model for vaccine development. We present a structural analysis of the stability of recombinant 18 kDa-hsp during different processing steps. Circular dichroism and ELISA were used to monitor protein structure after thermal stress, lyophilization and chemical modification. We observed that the 18 kDa-hsp is extremely resistant to a wide range of temperatures (60% of activity is retained at 80 degrees C for 20 min). N-Acylation increased its ordered structure by 4% and decreased its beta-T1 structure by 2%. ELISA demonstrated that the native conformation of the 18 kDa-hsp was preserved after hydrophobic modification by acylation. The recombinant 18 kDa-hsp resists to a wide range of temperatures and chemical modifications without loss of its main characteristic, which is to be a source of T epitopes. This resistance is probably directly related to its lack of organization at the level of tertiary and secondary structures.

Infrared and fluorescence spectroscopy for monitoring protein structure and interaction changes during cheese ripening

Sixteen experimental semi-hard cheeses, varying in moisture (42.1 to 49.8%), protein (20.2 to 25.9%) and fat (23.7 to 31.1%) content, were manufactured and ripened under controlled conditions. Fluorescence (tryptophan) and mid-infrared (Amide I and II regions) spectra were col- lected at 1, 21, 51 and 81 days of ripening in order to test the ability of spectroscopy to highlight the molecular changes that occur during this process. The mid-infrared and fluorescence spectral data from the experimental cheeses were analysed firstly by principal component analysis. Secondly, the correlations between the chemical domain and the spectral domains were studied by canonical corre- lation analysis methods. These analyses showed that each spectroscopic technique provided relevant information related to the cheese protein structure, which was used to discriminate each ripening stage. In addition, some spectral characteristics of ripened cheeses, linked to the initial chemical com- position and the initial protein network structure, were detected at the early stage of ripening. Finally, a canonical correlation analysis between the two sets of spectroscopic data was performed and al- lowed to clearly discriminate each stage of ripening and each cheese at the 4 ripening stages. A mo- lecular interpretation of these results involving the modifications of proteins, minerals and water interactions during ripening was attempted. This result demonstrated the interest of coupling two complementary spectroscopic techniques. Such coupling allowed the description of global character- istics of the investigated samples, which can be used for their characterisation. cheese / ripening / protein / structure / spectroscopy