Fourier transform infrared spectroscopic analysis of protein secondary structures

Infrared spectroscopy is one of the oldest and well established experimental techniques for the analysis of secondary structure of polypeptides and proteins. It is convenient, non-destructive, requires less sample preparation, and can be used under a wide variety of conditions. This review introduces the recent developments in Fourier transform infrared （FTIR） spectroscopy technique and its applications to protein structural studies. The experimental skills, data analysis, and correlations between the FTIR spectroscopic bands and protein secondary structure components are discussed. The applications of FTIR to the second- ary structure analysis, conformational changes, structural dynamics and stability studies of proteins are also discussed.     

Applications of ATR-FTIR spectroscopic imaging to biomedical samples

FTIR spectroscopic imaging in ATR (Attenuated Total Reflection) mode is a powerful tool for studying biomedical samples. This paper summarises recent advances in the applications of ATR-FTIR imaging to dissolution of pharmaceutical formulations and drug release. The use of two different ATR accessories to obtain chemical images of formulations in contact with water as a function of time is demonstrated. The innovative use of the diamond ATR accessory allowed in situ imaging of tablet compaction and dissolution. ATR-FTIR imaging was also applied to obtain images of the surface of skin and the spatial distribution of protein and lipid rich domains was obtained. Chemical images of cross-section of rabbit aorta were obtained using a diamond ATR accessory and the possibility of in situ imaging of arterial samples in contact with aqueous solution was demonstrated for the first time. This experiment opens an opportunity to image arterial samples in contact with solutions containing drug molecules. This approach may help in understanding the mechanisms of treatment of atherosclerosis.     

Applications of ATR-FTIR spectroscopic imaging to biomedical samples

FTIR spectroscopic imaging in ATR (Attenuated Total Reflection) mode is a powerful tool for studying biomedical samples. This paper summarises recent advances in the applications of ATR-FTIR imaging to dissolution of pharmaceutical formulations and drug release. The use of two different ATR accessories to obtain chemical images of formulations in contact with water as a function of time is demonstrated. The innovative use of the diamond ATR accessory allowed in situ imaging of tablet compaction and dissolution. ATR-FTIR imaging was also applied to obtain images of the surface of skin and the spatial distribution of protein and lipid rich domains was obtained. Chemical images of cross-section of rabbit aorta were obtained using a diamond ATR accessory and the possibility of in situ imaging of arterial samples in contact with aqueous solution was demonstrated for the first time. This experiment opens an opportunity to image arterial samples in contact with solutions containing drug molecules. This approach may help in understanding the mechanisms of treatment of atherosclerosis.     

Infrared spectroscopic imaging: Label-free biochemical analysis of stroma and tissue fibrosis

Infrared spectroscopic tissue imaging is a potentially powerful adjunct tool to current histopathology techniques. By coupling the biochemical signature obtained through infrared spectroscopy to the spatial information offered by microscopy, this technique can selectively analyze the chemical composition of different features of unlabeled, unstained tissue sections. In the past, the tissue features that have received the most interest were parenchymal and epithelial cells, chiefly due to their involvement in dysplasia and progression to carcinoma; however, the field has recently turned its focus toward stroma and areas of fibrotic change. These components of tissue present an untapped source of biochemical information that can shed light on many diverse disease processes, and potentially hold useful predictive markers for these same pathologies. Here we review the recent applications of infrared spectroscopic imaging to stromal and fibrotic regions of diseased tissue, and explore the potential of this technique to advance current capabilities for tissue analysis.     

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.     

Spectroscopic imaging of arteries and atherosclerotic plaques

Fourier transform infrared (FTIR) spectroscopic imaging using a focal plane array detector has been used to study atherosclerotic arteries with a spatial resolution of 3-4 microm, i.e., at a level that is comparable with cellular dimensions. Such high spatial resolution is made possible using a micro-attenuated total reflection (ATR) germanium objective with a high refractive index and therefore high numerical aperture. This micro-ATR approach has enabled small structures within the vessel wall to be imaged for the first time by FTIR. Structures observed include the elastic lamellae of the tunica media and a heterogeneous distribution of small clusters of cholesterol esters within an atherosclerotic lesion, which may correspond to foam cells. A macro-ATR imaging method was also applied, which involves the use of a diamond macro-ATR accessory. This study of atherosclerosis is presented as an illustrative example of the wider potential of these ATR imaging approaches for cardiovascular medicine and biomedical applications.     

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.     

Advances of infrared spectroscopy in natural product research

Natural product's properties are related to certain classes of compounds such as alkaloids, flavonoids, essential oils and others. Traditionally, separation techniques including thin layer chromatography (TLC), liquid chromatography (LC), gas chromatography (GC) and capillary electrophoresis (CE) even hyphenated to mass spectrometry (MS) were used for the elucidation, qualitative and quantitative analysis of individual compounds.In food industry, spectroscopic investigations using infrared radiation have been used to monitor and evaluate the composition and quality already since the early sixties. During the last four decades near-infrared spectroscopy (NIR; 800–2500 nm; 12,500–4000 cm⁻¹) has become one of the most attractive and used methods for analysis for the following reasons: it represents a non-invasive analytical tool allowing a fast and simultaneous qualitative and quantitative characterization of natural products and their constituents. Additionally, the development of custom-made hand-held instruments enables in-field measurement for determining the optimum harvest time.Attenuated total reflection (ATR) and Fourier transform infrared (FTIR) spectroscopic imaging are suitable not only for the differentiation of different plant species, but also to distinct various ingredients within a plant. FTIR spectroscopic microscopy enables molecular imaging of complex botanical samples and therefore the detection and characterization of the molecular components of biological tissue.In the present contribution, the principle, technique and methodology of the different infrared spectroscopic methods are described followed by a discussion of quantitative and qualitative application possibilities in the field of natural product analysis.     

Recent advances using green and red fluorescent protein variants

Fluorescent proteins have proven to be excellent tools for live-cell imaging. In addition to green fluorescent protein (GFP) and its variants, recent progress has led to the development of monomeric red fluorescent proteins (mRFPs) that show improved properties with respect to maturation, brightness, and the monomeric state. This review considers green and red spectral variants, their paired use for live-cell imaging in vivo, in vitro, and in fluorescence resonance energy transfer (FRET) studies, in addition to other recent “two-color” advances including photoswitching and bimolecular fluorescence complementation (BiFC). It will be seen that green and red fluorescent proteins now exist with nearly ideal properties for dual-color microscopy and FRET.     

Infrared spectroscopic analysis of tumor pathology

Infrared spectra of normal and malignant breast tissues were measured in the 600 cm(-1) to 4000 cm(-1) region. The measured spectroscopic features which are the spectroscopic fingerprints of the tissues contain the vital information about the malignant and normal tissues. Fourier Transform Infrared (FTIR) data on 25 cases of infiterating ductal carcinoma of breast with different grades of malignancy from patients of different age groups were analyzed. The samples were taken from the tumor sections of the tissue removed during surgery. Infrared spectra demonstrate significant spectral differences between the normal and the cancerous breast tissues. In particular changes in frequency and intensity in the spectra of protein, nucleic acid and glycogen vibrational modes as well as the band intensity ratios for lipid/proteins, protein/nucleic acids, protein/glycogen were observed. This allows to make a qualitative and semi quantitative evaluation of the changes in proliferation activity from normal to diseased tissue. It was evident that the sample to sample or patient to patient variations were small and the spectral differences between normal and diseased tissues were reproducible. The findings establish a framework for additional studies, which may enable us to establish a relation of the diseased state with its infrared spectra.     

Fourier transform infrared spectroscopy in high-pressure studies on proteins

Several aspects of the application of Fourier transform infrared spectroscopy (FTIR) in high-pressure studies on proteins are reviewed. Basic methodological considerations regarding spectral band assignments, quantitative analysis, and choice of pressure calibrants are also placed within the scope of this paper. This work attempts to evaluate recent developments in the field of high-pressure FTIR of proteins and its prospects for future. Particular attention is paid to the phenomenon of protein aggregation.     

Activity-based protein profiling in bacteria: Applications for identification of therapeutic targets and characterization of microbial communities

Activity-based protein profiling (ABPP) is a robust chemoproteomic technique that uses activity-based probes to globally measure endogenous enzymatic activity in complex proteomes. It has been utilized extensively to characterize human disease states and identify druggable targets in diverse disease conditions. ABPP has also recently found applications in microbiology. This includes using activity-based probes (ABPs) for functional studies of pathogenic bacteria as well as complex communities within a microbiome. This review will focus on recent advances in the use of ABPs to profile enzyme activity in disease models, screen for selective inhibitors of key enzymes, and develop imaging tools to better understand the host–bacterial interface.     

Automatic amide I frequency selection for rapid quantification of protein secondary structure from Fourier transform infrared spectra of proteins

Here we report the development of a new neural network based approach for rapid quantification of protein secondary structure from Fourier transform infrared (FTIR) spectra of proteins. A technique for efficiently reducing the amount of spectral data by almost 90% is suggested to facilitate faster neural network analysis. Additionally, an automatic procedure is introduced for selecting only those regions within the amide I band of protein FTIR spectra, which can be best related to secondary structure contents by subsequent neural network analysis. Based on a given reference set of FTIR spectra from proteins with known secondary structure, a subset of merely 29 out of 101 amide I absorbance values could be identified, which lead to an improved prediction accuracy. The average prediction accuracy achieved for helix, sheet, turn, bend, and other is 4.96% which is better than that achieved by alternative methods that have been previously reported indicating the significant potential of this approach. Our suggested automatic amide I frequency selection procedure may be easily extended to identify promising regions from spectral data recorded by other spectroscopic techniques, like for example circular dichroism spectroscopy.     

Spectroscopic methods for analysis of protein secondary structure

Several methods for determination of the secondary structure of proteins by spectroscopic measurements are reviewed. Circular dichroism (CD) spectroscopy provides rapid determinations of protein secondary structure with dilute solutions and a way to rapidly assess conformational changes resulting from addition of ligands. Both CD and Raman spectroscopies are particularly useful for measurements over a range of temperatures. Infrared (IR) and Raman spectroscopy require only small volumes of protein solution. The frequencies of amide bands are analyzed to determine the distribution of secondary structures in proteins. NMR chemical shifts may also be used to determine the positions of secondary structure within the primary sequence of a protein. However, the chemical shifts must first be assigned to particular residues, making the technique considerably slower than the optical methods. These data, together with sophisticated molecular modeling techniques, allow for refinement of protein structural models as well as rapid assessment of conformational changes resulting from ligand binding or macromolecular interactions. A selected number of examples are given to illustrate the power of the techniques in applications of biological interest.     

Interaction of Catechu Dye with DNA: Spectroscopic and In Silico Approach

Catechin, a yellow colored molecule obtained from the wood of Acacia catechu was analyzed for its interaction with synthetic DNA duplexes using spectroscopic analysis. UV-Visible spectroscopic analysis revealed the non-intercalative binding mode. Fourier Transform Infrared spectroscopy (FTIR) analysis expose chemical shift indicated by various vibrational stretches and an increase in the intensity of base stacking was observed by Circular Dichroism (CD), respectively. This inference was further confirmed through nuclear staining technique and also in electrophoretic technique; the dye quenches the fluorescent intensity of ethidium bromide. The result of fluorescence spectroscopy was in concordance with the electrophoretic technique. In addition, the spectroscopic results were in accordance with the molecular docking studies of specific catechin compound from the catechu dye with CT-DNA. This kind of site specificity is a gain in the medicinal field as the drug can be DNA targeted for cancer therapeutics. The present work reveals that catechu dye has a noteworthy application in the field of medical bioscience.     

Discrimination of single‐porin Escherichia (E.) coli mutants by ATR and transmission mode FTIR spectroscopy

Vibrational spectroscopy has long been used in bacterial identification with different levels of taxonomic discrimination but its true potential for intra‐species differentiation remains poorly explored. Herein, both transmission Fourier‐transform infrared (FTIR) and attenuated total reflectance (ATR)‐FTIR spectroscopy are used to analyse E. coli strains that differ solely in their porin expression profile. In this previously unreported approach, the applicability of both FTIR‐spectroscopy techniques is compared with the same collection of unique strains. ATR‐FTIR spectroscopy proved to reliably distinguish between several E. coli porin mutants with an accuracy not replicated by FTIR in transmission mode (using previously optimized procedures). Further studies should allow the identification of the individual contribution of the single porin channel to the overall bacterial infrared spectrum and of molecular predictive patterns of porin alterations. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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.     

Quantitative imaging of protein interactions in the cell nucleus

Over the past decade, genetically encoded fluorescent proteins have become widely used as noninvasive markers in living cells. The development of fluorescent proteins, coupled with advances in digital imaging, has led to the rapid evolution of live-cell imaging methods. These approaches are being applied to address biological questions of the recruitment, co-localization, and interactions of specific proteins within particular subcellular compartments. In the wake of this rapid progress, however, come important issues associated with the acquisition and analysis of ever larger and more complex digital imaging data sets. Using protein localization in the mammalian cell nucleus as an example, we will review some recent developments in the application of quantitative imaging to analyze subcellular distribution and co-localization of proteins in populations of living cells. In this report, we review the principles of acquiring fluorescence resonance energy transfer (FRET) microscopy measurements to define the spatial relationships between proteins. We then discuss how fluorescence lifetime imaging microscopy (FLIM) provides a method that is independent of intensity-based measurements to detect localized protein interactions with spatial resolution. Finally, we consider potential problems associated with the expression of proteins fused to fluorescent proteins for FRET-based measurements from living cells.     

Fourier transform infrared microspectroscopy identifies early lineage commitment in differentiating human embryonic stem cells

Human ESCs (hESCs) are a valuable tool for the study of early human development and represent a source of normal differentiated cells for pharmaceutical and biotechnology applications and ultimately for cell replacement therapies. For all applications, it will be necessary to develop assays to validate the efficacy of hESC differentiation. We explored the capacity for FTIR spectroscopy, a technique that rapidly characterises cellular macromolecular composition, to discriminate mesendoderm or ectoderm committed cells from undifferentiated hESCs. Distinct infrared spectroscopic “signatures” readily distinguished hESCs from these early differentiated progeny, with bioinformatic models able to correctly classify over 97% of spectra. These data identify a role for FTIR spectroscopy as a new modality to complement conventional analyses of hESCs and their derivatives. FTIR spectroscopy has the potential to provide low-cost, automatable measurements for the quality control of stem and differentiated cells to be used in industry and regenerative medicine.