Recent applications of ATR FTIR spectroscopy and imaging to proteins

Attenuated Total Reflection (ATR) Fourier Transform Infrared (FTIR) spectroscopy is a label-free, non-destructive analytical technique that can be used extensively to study a wide variety of different molecules in a range of different conditions. The aim of this review is to discuss and highlight the recent advances in the applications of ATR FTIR spectroscopic imaging to proteins. It briefly covers the basic principles of ATR FTIR spectroscopy and ATR FTIR spectroscopic imaging as well as their advantages to the study of proteins compared to other techniques and other forms of FTIR spectroscopy. It will then go on to examine the advances that have been made within the field over the last several years, particularly the use of ATR FTIR spectroscopy for the understanding and development of protein interaction with surfaces. Additionally, the growing potential of Surface Enhanced Infrared Spectroscopy (SEIRAS) within this area of applications will be discussed. The review includes the applications of ATR FTIR imaging to protein crystallisation and for high-throughput studies, highlighting the future potential of the technology within the field of protein structural studies and beyond.     

Secondary structures of proteins adsorbed onto aluminum hydroxide: infrared spectroscopic analysis of proteins from low solution concentrations

Comparative studies of the secondary structures of six model proteins, adsorbed onto aluminum hydroxide gel (Alhydrogel) or in aqueous solution, were carried out by Fourier transform infrared (FTIR) spectroscopy. The analysis of high-quality spectra of all six model proteins, with a broad range of secondary structure compositions, obtained at 15 mg/ml by the conventional method and at 0.5 and 1.0 mg/ml adsorbed to Alhydrogel revealed that adsorption onto hydrophilic surfaces of aluminum hydroxide particles did not alter the secondary structures of the proteins. The results of this study suggest that adsorbing proteins to Alhydrogel provides a means of obtaining FTIR spectra to study secondary structure and conformational changes of proteins in aqueous solution at very low concentrations. The new procedure effectively lowers the concentration requirement for FTIR studies of proteins in aqueous solutions by at least 40-fold, as compared with the conventional FTIR method. It permits FTIR study of proteins to be carried out in the same concentration range as is used for circular dichroism and fluorescence, thereby making it possible to compare structural information obtained by three commonly used techniques in protein biophysical characterization.     

Application of ATR‐FTIR spectroscopy to the study of thermally induced changes in secondary structure of protein molecules in solid state

FTIR spectroscopy in combination with ATR sampling technique is the most accessible analytical technique to study secondary structure of proteins both in solid and aqueous solution. Although several studies have demonstrated the applications of ATR‐FTIR to study conformational changes of solid dried proteins due to dehydration, there are no reports that demonstrate the application of ATR‐FTIR in the study of thermally induced changes of secondary structure of biomolecules directly on the solid state. In this study, four biomolecules of pharmaceutical interest, lysozyme, myoglobine, chymotripsin and human growth hormone (hGH), were studied on the solid state before and after different thermal treatments in order to relate changes of secondary structure to partial or total thermal denaturation processes. The results obtained provide experimental evidence that protein thermal denaturation in the solid state can be detected by displacement of carbonyl bands which correspond to conformational transformations between α–helix to β‐sheet or intermolecular β‐sheet; the molecules studied undergo this transformation when exposed to a temperature close to their denaturation temperature which may become irreversible depending on the extent of the heating treatment. These findings demonstrate that ATR‐FTIR is an effective and time efficient technique that allows the monitoring of the protein thermal denaturation process of solid samples without further reconstitution or prior sample preparation. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 574–584, 2015.     

振动光谱与蛋白质二级结构

红外光谱和拉曼光谱(统称分子振动光谱)应用于蛋白质构象研究经历了定性阶段和半定量阶段.近5年来,去卷积和二阶导教等提高分辨率方法以及曲线拟合方法的应用,使得振动光谱在定量计算蛋白质二级结构和监测蛋白质构象变化等方面取得突破性的进展,进入了定量化的阶段.     

Total chemical synthesis of fully functional Photoactive Yellow Protein

The Photoactive Yellow Protein (PYP) is a structural prototype for the PAS superfamily of proteins, which includes hundreds of receptor and regulatory proteins from all three kingdoms of life. PYP itself is a small globular protein that undergoes a photocycle involving a series of conformational changes in response to light excitation of its p-coumaric acid chromophore, making it an excellent model system to study the molecular basis of signaling in the PAS super family. To enable novel chemical approaches to elucidating the structural changes that accompany signaling in PYP, we have chemically synthesized the 125 amino acid residue protein molecule using a combination of Boc chemistry solid phase peptide synthesis and native chemical ligation. Synthetic PYP exhibits the wildtype photocycle, as determined in photobleaching studies. Planned future studies include incorporation of site-specific isotopic labels into specific secondary structural elements to determine which structural elements are involved in signaling state formation using difference FTIR spectroscopy.     

Neuro-fuzzy structural classification of proteins for improved protein secondary structure prediction

Fourier transform infrared (FTIR) spectroscopy is a very flexible technique for characterization of protein secondary structure. Measurements can be carried out rapidly in a number of different environments based on only small quantities of proteins. For this technique to become more widely used for protein secondary structure characterization, however, further developments in methods to accurately quantify protein secondary structure are necessary. Here we propose a structural classification of proteins (SCOP) class specialized neural networks architecture combining an adaptive neuro-fuzzy inference system (ANFIS) with SCOP class specialized backpropagation neural networks for improved protein secondary structure prediction. Our study shows that proteins can be accurately classified into two main classes "all alpha proteins" and "all beta proteins" merely based on the amide I band maximum position of their FTIR spectra. ANFIS is employed to perform the classification task to demonstrate the potential of this architecture with moderately complex problems. Based on studies using a reference set of 17 proteins and an evaluation set of 4 proteins, improved predictions were achieved compared to a conventional neural network approach, where structure specialized neural networks are trained based on protein spectra of both "all alpha" and "all beta" proteins. The standard errors of prediction (SEPs) in % structure were improved by 4.05% for helix structure, by 5.91% for sheet structure, by 2.68% for turn structure, and by 2.15% for bend structure. For other structure, an increase of SEP by 2.43% was observed. Those results were confirmed by a "leave-one-out" run with the combined set of 21 FTIR spectra of proteins.     

Towards developing a protein infrared spectra databank (PISD) for proteomics research

Fourier transform infrared (FTIR) spectroscopy is an attractive tool for proteomics research as it can be used to rapidly characterize protein secondary structure in aqueous solution. Recently, a number of secondary structure prediction methods based on reference sets of FTIR spectra from proteins with known structure from X-ray crystallography have been suggested. These prediction methods, often referred to as pattern recognition based approaches, demonstrated good prediction accuracy using some error measure, e.g., the standard error of prediction (SEP). However, to avoid possible adverse effects from differences in recording, the analysis has been mostly based on reference sets of FTIR spectra from proteins recorded in one laboratory only. As a result, these studies were based on reference sets of FTIR spectra from a limited number of proteins. Pattern recognition based approaches, however, rely on reference sets of FTIR spectra from as many proteins as possible representing all possible band shape variation to be related to the diversity of protein structural classes. Hence, if we want to build reliable pattern recognition based systems to support proteomics research, which are capable of making good predictions from spectral data of any unknown protein, one common goal should be to build a comprehensive protein infrared spectra databank (PISD) containing FTIR spectra of proteins of known structure. We have started the process of developing a comprehensive PISD composed of spectra recorded in different laboratories. As part of this work, here we investigate possible effects on prediction accuracy achieved by a neural network analysis when using reference sets composed of FTIR spectra from different laboratories. Surprisingly low magnitude of difference in SEPs throughout all our experiments suggests that FTIR spectra recorded in different laboratories may be safely combined into one reference set with only minor deterioration of prediction accuracy in the worst case.     

Conformation and orientation of the gene 9 minor coat protein of bacteriophage M13 in phospholipid bilayers

The membrane-bound state of the gene 9 minor coat protein of bacteriophage M13 was studied in model membrane systems, which varied in lipid head group and lipid acyl chain composition. By using FTIR spectroscopy and subsequent band analysis a quantitative analysis of the secondary structure of the protein was obtained. The secondary structure of the gene 9 protein predominantly consists of alpha-helical (67%) and turn (33%) structures. The turn structure is likely to be located C-terminally where it has a function in recognizing the phage DNA during bacteriophage assembly. Attenuated total reflection FTIR spectroscopy was used to determine the orientation of gene 9 protein in the membrane, revealing that the alpha-helical domain is mainly transmembrane. The conformational and orientational measurements result in two models for the gene 9 protein in the membrane: a single transmembrane helix model and a two-helix model consisting of a 15 amino acid long transmembrane helix and a 10 amino acid long helix oriented parallel to the membrane plane. Potential structural consequences for both models are discussed.     

Methodological advances and strategies for high resolution structure determination of cellular protein aggregates

Aggregation of proteins is at the nexus of molecular processes crucial to aging, disease, and employing proteins for biotechnology and medical applications. There has been much recent progress in determining the structural features of protein aggregates that form in cells; yet, owing to prevalent heterogeneity in aggregation, many aspects remain obscure and often experimentally intractable to define. Here, we review recent results of structural studies for cell-derived aggregates of normally globular proteins, with a focus on high-resolution methods for their analysis and prediction. Complementary results obtained by solid-state NMR spectroscopy, FTIR spectroscopy and microspectroscopy, cryo-EM, and amide hydrogen/deuterium exchange measured by NMR and mass spectrometry, applied to bacterial inclusion bodies and disease inclusions, are uncovering novel information on in-cell aggregation patterns as well as great diversity in the structural features of useful and aberrant protein aggregates. Using these advances as a guide, this review aims to advise the reader on which combination of approaches may be the most appropriate to apply to their unique system.     

Overestimated accuracy of circular dichroism in determining protein secondary structure

Circular dichroism (CD) is a spectroscopic technique widely used for estimating protein secondary structures in aqueous solution, but its accuracy has been doubted in recent work. In the present paper, the contents of nine globular proteins with known secondary structures were determined by CD spectroscopy and Fourier transform infrared spectroscopy (FTIR) in aqueous solution. A large deviation was found between the CD spectra and X-ray data, even when the experimental conditions were optimized. The content determined by FTIR was in good agreement with the X-ray crystallography data. Therefore, CD spectra are not recommended for directly calculating the content of a protein’s secondary structure.     

FTIR difference spectroscopy of bacteriorhodopsin: Toward a molecular model

Bacteriorhodopsin (bR) is a light-driven proton pump whose function includes two key membrane-based processes, active transport and energy transduction. Despite extensive research on bR and other membrane proteins, these processes are not fully understood on the molecular level. In the past ten years, the introduction of Fourier transform infrared (FTIR) difference spectroscopy along with related techniques including time-resolved FTIR difference spectroscopy, polarized FTIR, and attenuated total reflection FTIR has provided a new approach for studying these processes. A key step has been the utilization of site-directed mutagenesis to assign bands in the FTIR difference spectrum to the vibrations of individual amino acid residues. On this basis, detailed information has been obtained about structural changes involving the retinylidene chromophore and protein during the bR photocycle. This includes a determination of the protonation state of the four membrane-embedded Asp residues, identification of specific structurally active amino acid residues, and the detection of protein secondary structural changes. This information is being used to develop an increasingly detailed picture of the bR proton pump mechanism.     

A comparative infrared spectroscopic study of glycoside hydrolases from extremophilic archaea revealed different molecular mechanisms of adaptation to high temperatures

The identification of the determinants of protein thermal stabilization is often pursued by comparing enzymes from hyperthermophiles with their mesophilic counterparts while direct structural comparisons among proteins and enzymes from hyperthermophiles are rather uncommon. Here, oligomeric beta-glycosidases from the hyperthermophilic archaea Sulfolobus solfataricus (Ss beta-gly), Thermosphaera aggregans (Ta beta-gly), and Pyrococcus furiosus (Pf beta-gly), have been compared. Studies of FTIR spectroscopy and kinetics of thermal inactivation showed that the three enzymes had similar secondary structure composition, but Ss beta-gly and Ta beta-gly (temperatures of melting 98.1 and 98.4 degrees C, respectively) were less stable than Pf beta-gly, which maintained its secondary structure even at 99.5 degrees C. The thermal denaturation of Pf beta-gly, followed in the presence of SDS, suggested that this enzyme is stabilized by hydrophobic interactions. A detailed inspection of the 3D-structures of these enzymes supported the experimental results: Ss beta-gly and Ta beta-gly are stabilized by a combination of ion-pairs networks and intrasubunit S-S bridges while the increased stability of Pf beta-gly resides in a more compact protein core. The different strategies of protein stabilization give experimental support to recent theories on thermophilic adaptation and suggest that different stabilization strategies could have been adopted among archaea.     

Downscaling Fourier transform infrared spectroscopy to the micrometer and nanogram scale: secondary structure of serotonin and acetylcholine receptors

High signal-to-noise Fourier transform infrared (FTIR) spectra of the 5-hydroxytryptamine (serotonin) receptor (5-HT(3)R) and the nicotinic acetylcholine receptor (nAChR) were obtained by microscope FTIR spectroscopy using micrometer-sized, fully hydrated protein films. Because this novel procedure requires only nanogram quantities of membrane proteins, which is 4-5 orders of magnitude less than the amount of protein typically used for conventional FTIR spectroscopy, it opens the possibility to access the structure and dynamics of many important mammalian receptor proteins. The secondary structure of detergent-solubilized 5-HT(3)R determined by curve fitting of the amide I band yielded 36% alpha-helix, 33% beta-strand, 15% beta-turn, and 16% nonregular structures, which remained unchanged upon reconstitution in lipid membranes. From hydrogen-deuterium exchange, the secondary structure of the water-accessible part of 5-HT(3)R was determined as 14% alpha-helix, 16% beta-strand, 26% beta-turn, and 14% nonregular structures. Interestingly, we found that both the overall and the water-accessible nAChR secondary structures were nearly identical to those of 5-HT(3)R, in agreement with predicted structures of this class of receptors. This is the first time that structural investigations were obtained for two closely related ligand-gated ion channels under strictly identical experimental conditions.     

Site-specific examination of secondary structure and orientation determination in membrane proteins: the peptidic (13)C=(18)O group as a novel infrared probe

Detailed site-specific information can be exceptionally useful in structural studies of macromolecules in general and proteins in particular. Such information is usually obtained from spectroscopic studies using a label/probe that can reflect on particular properties of the protein. A suitable probe must not modify the native properties of the protein, and should yield interpretable structural information, as is the case with isotopic labels used by Fourier transform infrared (FTIR) spectroscopy. In particular, 1-(13)C=(18)O labels have been shown to relay site-specific secondary structure and orientational information, although limited to small peptides. The reason for this limitation is the high natural abundance of (13)C and the lack of baseline resolution between the main amide I band and the isotope-edited peak. Herein, we dramatically extend the utility of isotope edited FTIR spectroscopy to proteins of virtually any size through the use of a new 1-(13)C=(18)O label. The double-isotope label virtually eliminates any contribution from natural abundance (13)C. More importantly, the isotope-edited peak is further red-shifted (in accordance with ab initio Hartree-Fock calculations) and is now completely baseline resolved from the main amide I band. Taken together, this new label enables determination of site specific secondary structure and orientation in proteins of virtually any size. Even in small peptides 1-(13)C=(18)O is far preferable as a label in comparison to 1-(13)C=(18)O since it enables analysis without the need for any deconvolution or peak fitting procedures. Finally, the results obtained herein represent the first stage in the application of site-directed dichroism to the structural elucidation of polytopic membrane proteins.     

Application of docking methodologies to modeled proteins

Protein docking is essential for structural characterization of protein interactions. Besides providing the structure of protein complexes, modeling of proteins and their complexes is important for understanding the fundamental principles and specific aspects of protein interactions. The accuracy of protein modeling, in general, is still less than that of the experimental approaches. Thus, it is important to investigate the applicability of docking techniques to modeled proteins. We present new comprehensive benchmark sets of protein models for the development and validation of protein docking, as well as a systematic assessment of free and template-based docking techniques on these sets. As opposed to previous studies, the benchmark sets reflect the real case modeling/docking scenario where the accuracy of the models is assessed by the modeling procedure, without reference to the native structure (which would be unknown in practical applications). We also expanded the analysis to include docking of protein pairs where proteins have different structural accuracy. The results show that, in general, the template-based docking is less sensitive to the structural inaccuracies of the models than the free docking. The near-native docking poses generated by the template-based approach, typically, also have higher ranks than those produces by the free docking (although the free docking is indispensable in modeling the multiplicity of protein interactions in a crowded cellular environment). The results show that docking techniques are applicable to protein models in a broad range of modeling accuracy. The study provides clear guidelines for practical applications of docking to protein models.     

Vibrational circular dichroism studies of epidermal growth factor and basic fibroblast growth factor.

Vibrational circular dichroism (VCD) studies are reported for two unrelated recombinant growth factor proteins: epidermal growth factor and basic fibroblast growth factor (bFGF). NMR, electronic CD, and bFGF X-ray studies indicate that these two proteins are primarily composed of beta-sheet and loop secondary structure elements with no detectable alpha-helices. Two reports on solution conformation of these proteins using FTIR absorption spectroscopy with subsequent resolution enhancement confirmed the presence of a large fraction of a beta-sheet conformation but in addition indicated the presence of large absorption bands in the 1650-1656 cm-1 region, which are typically assigned to alpha-helices. The VCD spectra of both proteins have band shapes that strongly resemble those of other high beta-sheet fraction proteins, such as the trypsin family of proteins. Quantitative analysis of the VCD spectra also indicates that these proteins are predominantly in beta-sheet and extended ("other") conformations with very little alpha-helix fraction. These results agree with the CD interpretation and affirm that the FTIR peaks in the region 1650-1656 cm-1 can be assigned to loops. This study provides an example of the limitations of using FTIR frequencies alone for examination of protein secondary structure.     

Application of multivariate curve resolution-alternating least squares (MCR-ALS) for secondary structure resolving of proteins

Fourier transform infrared (FTIR) spectroscopy is the most common spectroscopic technique used for study of protein structure. Initially, band deconvolution techniques were applied to determine the secondary structure of proteins. Recently, several multivariate regression methods have been used to predict the secondary structure of proteins as an alternative to the previous methods. Multivariate curve resolution-alternating least squares (MCR-ALS) was applied on the FTIR spectra of proteins to resolve the fraction and spectral profiles of different structural motifs. Initial estimates of spectral profiles of different protein motifs were built using orthogonal projection approach (OPA). Predicted fractions of α-helix and β-sheet obtained by MCR-ALS technique were compared with those from partial least squares (PLS) modeling which revealed superiority of the former. If we consider the possibility of pure spectra prediction in addition to the prediction of secondary structure from the data set, MCR-ALS can be proposed as a very valuable alternative for qualitative and quantitative study of protein structures.     

Multidimensional NMR methods for protein structure determination

Structural studies of proteins are critical for understanding biological processes at the molecular level. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for obtaining structural and dynamic information on proteins and protein-ligand complexes. In the present review, methodologies for NMR structure determination of proteins and macromolecular complexes are described. In addition, a number of recent advances that reduce the molecular weight limitations previously imposed on NMR studies of biomolecules are discussed, highlighting applications of these technologies to protein systems studied in our laboratories.     

Binding of glutamine to glutamine-binding protein from Escherichia coli induces changes in protein structure and increases protein stability

Glutamine-binding protein (GlnBP) from Escherichia coli is a monomeric protein localized in the periplasmic space of the bacterium. It is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane. The protein consists of two similar globular domains linked by two peptide hinges, and X-ray crystallographic data indicate that the two domains undergo large movements upon ligand binding. Fourier transform infrared spectroscopy (FTIR) was used to analyze the structure and thermal stability of the protein in detail. The data indicate that glutamine binding induces small changes in the secondary structure of the protein and that it renders the structure more thermostable and less flexible. Detailed analyses of IR spectra show a lower thermal sensitivity of alpha-helices than beta-sheets in the protein both in the absence and in the presence of glutamine. Generalized two-dimensional (2D) analyses of IR spectra reveal the same sequence of unfolding events in the protein in the absence and in the presence of glutamine, indicating that the amino acid does not affect the unfolding pathway of the protein. The data give new insight into the structural characteristics of GlnBP that are useful for both basic knowledge and biotechnological applications.