Ebook: Biological and Biomedical Infrared Spectroscopy
Although infrared spectroscopy has been applied with success to the study of important biological and biomedical processes for many years, key advances in this vibrant technique have led to its increasing use, ranging from characterisation of individual macromolecules (DNA, RNA, lipids, proteins) to human tissues, cells and their components. Infrared spectroscopy thus has a significant role to play in the analysis of the vast number of genes and proteins being identified by the various genomic sequencing projects. Whilst this book gives an overview of the field it highlights more recent developments, such as the use of bright synchrotron radiation for recording infrared spectra, the development of two-dimensional infrared spectroscopy and the ability to record infrared spectra at ultrafast speeds. The main focus is on the mid-infrared region, since the great majority of studies are carried out in this region but there is increasing use of the near infrared for biomedical applications and a chapter is devoted to this part of the spectrum. Major advances in theoretical analysis have also enabled better interpretation of the infrared spectra of biological molecules and these are covered. The editors, Professor Andreas Barth of Stockholm University, Stockholm, Sweden and Dr Parvez I. Haris of De Montfort University, Leicester, U.K., who both have extensive research experience in biological infrared spectroscopy per se and in its use in the solution of biophysical problems, have felt it timely therefore to bring together this book. The book is intended for use both by research scientists already active in the use of biological infrared spectroscopy and for those coming new to the technique. Graduate students will also find it useful as an introduction to the technique.
This book aims to provide an insight into some of the key areas where infrared spectroscopy has been successfully applied to understand important biological and biomedical processes. It highlights the latest advances and the directions for the future. The book provides a historical framework for the development of biological infrared spectroscopy. Key methodologies that are in current use and latest advances, in both theoretical and practical aspects, are discussed. Examples of applications, ranging from characterisation of individual macromolecules (DNA, RNA, lipids, proteins) to complex systems such as human tissues, cells and whole organisms are covered. The main focus is in the mid-infrared region as the vast majority of studies are conducted in this region. However, there is increasing use of the near-infrared region for biomedical application and hence a chapter is devoted to this part of the infrared spectrum.
Biological spectroscopy is a highly interdisciplinary field of research requiring involvement of life scientists and analytical chemists. Advances in instrumentation technology and methods for analysis and interpretation of the spectroscopic data require input from multiple disciplines including Chemistry, Physics, Mathematics, Computer Science and Engineering. It is this cooperation between scientists from diverse disciplines that ultimately results in the utilisation of a physical technique for understanding the molecular details of biological processes and systems. Such cooperation is vital if spectroscopists are to play a significant role in the analysis of the vast number of genes and proteins that are being identified by the various genome sequencing projects. Currently, it is not impossible for a gene sequencing laboratory to produce as much data in less than a week as was produced by Shakespeare in his entire life-time. However, an understanding of the molecular details of the genes and proteins identified, and their diverse interactions, require application of biophysical techniques such as infrared spectroscopy. Continued technological development in spectroscopic methods is vital to keep pace with the breathtaking advances in the field of molecular biology.
Nearly 400 years ago Shakespeare described the “seven ages” of life in the following manner:
“All the world's a stage, And all the men and women merely players: They have their exits and their entrances; And one man in his time plays many parts, His acts being seven ages.”
Using this as an analogy, Laitinen in 1973 wrote an editorial in Analytical Chemistry describing the seven ages of an analytical method (H.A. Laitinen, Anal. Chem. 45 (1973) 2305). He used infrared spectroscopy as an example to illustrate how it has reached its “seventh age”. His description of this “seventh age” is as follows:
“Seventh, a period of senescence occurs as other methods of greater speed, economy, convenience, sensitivity, selectivity, etc., surpass the method under consideration.”
It is surprising that Laitinen chose infrared spectroscopy as his example, since at that time the first commercial Fourier transform infrared spectrometers were being delivered to laboratories around the world. As such it was a very exciting time for infrared spectroscopy. Indeed, a year earlier, in 1972, Peter Griffiths published a letter in the same journal entitled “Trading rules” in infrared Fourier transform spectroscopy" (P.R. Griffiths, Anal. Chem., 44 (1972), 1909). As an editor of the journal, Laitinen must have been aware of the revolution taking place in infrared spectroscopy. The widespread availability of FT instruments and the use of computers for recording and analysis of infrared spectra, heralded a new era in infrared spectroscopy. Now it was possible to analyse biological molecules, in aqueous media, at fast speeds and at high resolutions that was virtually impossible with dispersive instruments.
Far from reaching its “seventh age” infrared spectroscopy is a vibrant methodology playing a central role in some of the latest discoveries in biology and medicine, including some recent Nobel Prize winning work. For example, Stanley Prusiner was awarded the Nobel Prize for Physiology or Medicine in 1997 and infrared spectroscopy played an important role in his work. In a section of his Nobel lecture (S.B. Prusiner, Proc. Natl. Acad. Sci. USA, 95 (1998), 13363-13383) he states the following:
“For more than 25 years, it had been widely accepted that the amino acid sequence specifies one biologically active conformation of a protein…. Yet in scrapie we were faced with the possibility that one primary structure for PrP might adopt at least two different conformations to explain the existence of both PrPC and PrPSc. When the secondary structures of the PrP isoforms were compared by optical spectroscopy, they were found to be markedly different…. Fourier-transform infrared (FTIR) and circular dichroism (CD) studies showed that PrPC contains about 40% α-helix and little β-sheet, whereas PrPSc is composed of about 30% α-helix and 45% β-sheet…). Nevertheless, these two proteins have the same amino acid sequence!”
It is noteworthy that the abnormal form of the prion protein (PrPSc) misfolds and forms aggregates that are virtually impossible for characterisation using X-ray crystallography, NMR and CD spectroscopy. In order to overcome this problem, Prusiner and coworkers used infrared spectroscopy to obtain direct evidence for an increase in betasheet structure in the PrPSc aggregates.
In recent years infrared spectroscopy is going through a renaissance catalysed by some exciting developments in technology. This includes the use of the bright synchrotron radiation for recording infrared spectra. Latest breakthroughs also include the development of two-dimensional infrared spectroscopy and the ability to record infrared spectra at ultrafast speeds. There are also some major advances in theoretical analysis that is enabling a better interpretation of the infrared spectra of biological molecules. Considering these advances, we felt it would be timely to produce a book that brings together some of the key developments in the field. The book is intended for both experts and those who are new to the field of biological infrared spectroscopy. It would be particularly beneficial for graduate students and research scientists in both industry and academia.
Finally, we would like to thank all the authors who have contributed in this volume. Without their cooperation it would not have been possible to accomplish this task.
Andreas Barth (Stockholm University, Stockholm, Sweden), Parvez I. Haris (De Montfort University, Leicester, UK)
History of infrared spectroscopy as well as current technology and applications are reviewed.
Reaction-induced infrared difference spectroscopy of proteins is reviewed. This technique enables detailed characterization of enzyme function on the level of single bonds of proteins, cofactors or substrates. Discussed are methods to initiate protein reactions in the infrared samples, general aspects of spectra interpretation, measurements of enzyme activity and studies of protein function at the example of the Ca2+ pump.
The investigation of protein structural dynamics on short time scales (100 fs to 100 ps) using ultrafast two dimensional (2D-IR) vibrational echo spectroscopy is presented. Under thermal equilibrium conditions, a protein's structure is constantly fluctuating among conformations associated with different positions on the broad rough minimum on the free energy landscape. Although different conformational substates may be apparent in a vibrational absorption spectrum, linear IR absorption spectra cannot provide information on structural dynamics because dynamical information is masked by inhomogeneous broadening of the lineshapes. 2D-IR vibrational echo spectroscopy makes structural fluctuations a direct experimental observable. Changes in structure manifest themselves through the time evolution of the 2D-IR line shape (spectral diffusion). Here details of the experimental method including the pulse sequence, heterodyne detection to provide full phase information, and the extraction of the molecular dynamics from 2D-IR spectra are outlined. The method and the nature of the information that can be obtained are illustrated with four examples: the influence of mutations on myoglobin dynamics, differences between the dynamics of neuroglobin and myoglobin, the effect of the disulfide bond in neuroglobin on its structural dynamics, and how substrate binding to the enzyme horseradish peroxidase influences its structural fluctuations.
The information retrieved from FTIR spectra largely depends on both the quality of the original spectra and on the correction and processing methods. This contribution reviews the entire process driving to a fine and reliable interpretation of the data.
One of the major challenges of the post-genomic era is the rapid characterisation of protein structure. High-throughput structural genomic projects involving X-ray crystallography and NMR spectroscopy are in progress to solve the three-dimensional structures of a large of number of proteins. These techniques have their advantages and disadvantages and cannot be applied to study all proteins, giving sufficient opportunity for other techniques to also a play significant role in proteomics research. Fourier transform infrared (FTIR) spectroscopy is one of the techniques that has gained popularity in this area since measurements on small quantities of proteins can be carried out very rapidly in various environments. However, there is a need for improvements in the interpretation of protein FTIR infrared spectra and development of methods for accurately quantifying protein secondary structure from infrared spectra of proteins. Over the years, much progress has been made in this area and here we provide an overview of the major progress made so far, along with their strengths and weaknesses. The particular focus of the Chapter is on methods used for quantitative prediction of secondary structure from infrared spectra.
Vibrational spectra are frequently used for studies of the structure and dynamics of peptides and proteins. Structural interpretation of the experimental data, however, requires theoretical simulation of the spectra for model peptide geometries. Quantum mechanical, in particular density functional theory (DFT), methods have proven exceptionally valuable for calculations of the vibrational force fields and both IR and Raman intensities. A brief review of some recent trends in computation of molecular force fields and spectral intensities is presented. Particular attention is paid to experiments involving circularly polarized light, as these provide enhanced structural information for chiral molecules. Following a historical overview of common approaches, the fundamental theoretical aspects of the calculations of the molecular vibrational spectra are summarized. Special emphasis is given to the problem of simulating spectra for biological molecules (oligo-peptides and nucleotides, proteins and nucleic acids) using DFT methods. The methodology for simulations of large biopolymers with DFT level force fields and intensity parameters abstracted from smaller molecules is reviewed. Several examples with the discussion of successes and difficulties of the vibrational spectra simulations for model peptides are presented. Finally, methods for incorporating the solvent in the spectral simulations are reviewed and discussed.
Amide I vibrational spectra of proteins can provide critical information on secondary structure elements and structural inhomogeneity. Despite that there exist a number of linear and nonlinear vibrational spectroscopic studies reported, it has been quite difficult to quantitatively simulate the amide I vibrational spectra of polypeptides and proteins. To achieve this goal, we have developed theoretical and computational methods such as constrained molecular dynamics simulation, Hessian matrix reconstruction method, and fragmentation approximation to describe delocalized amide I normal modes of proteins as linear combinations of amide I local modes. By using the computational scheme, amide I IR, vibrational circular dichroism, and two-dimensional IR photon echo spectra of polypeptides and protein in solution were simulated and compared with experimental results. The structure-spectrum relationships established are discussed. It is believed that the present computational method will be of use in shedding light on the underlying vibrational dynamics of protein as well as in interpreting experimentally measured linear and nonlinear amide I spectra of proteins in the future.
Detailed analysis of molecular interactions is the key to understanding the mechanism of signal transduction and its intervention by inhibitory compounds. In this chapter, we shall review and discuss the application of isotope edited FTIR spectroscopy to the investigation of protein-protein interactions. Recently we have employed this technique to investigate the molecular interactions of granulocyte colony stimulating factor (G-CSF) with the isolated immunoglobular domain (Ig) of its receptor [1,2]. To resolve the amide I' band overlap of G-CSF with that of the receptor in the FTIR spectrum of the complex, 13C/15N uniformly labeled G-CSF was prepared for this study. By comparing the FTIR spectra of the isotope-labeled G-CSF and the isolated receptor with that of the complex, we have provided spectral evidence that the AB loop region involving the unique 310 helix segment of G-CSF likely undergoes a conformational change to a regular α-helix upon binding to the receptor domain. The IR data also indicate significant conformational changes involving β-turns and irregular structures in the Ig domain of the receptor in the complex. Furthermore, FTIR spectra of G-CSF, the receptor, and their complex demonstrate clearly that protein conformations of both G-CSF and the receptor are dramatically stabilized by complex formation. Together, the current data strongly suggest that the AB loop region including the 310 helix interacts specifically with the immunoglobulin-like domain of the receptor, which may play a role in receptor dimerization. This conclusion supports the structural model recently proposed by Layton and co-workers . In summary, this work demonstrates that specific structural information of protein-protein complexes can be obtained by employing isotope-edited FTIR spectroscopy.
This essay shows how Fourier Transform infrared spectroscopy (FTIR) can be applied to study membrane phase behavior of cells that are relevant for biomedical applications such as red blood cells and platelets. FTIR studies are minimally invasive and do not require labeling. FTIR can give unique information on conformation and stability of membranes in cells that are exposed to heating, freezing or dehydration stress. By combining in situ FTIR techniques with cell viability studies, cell damage can be correlated with membrane phase changes. Understanding the complex behavior of biomembranes during heating, freezing and drying is directly relevant for thermal processing of cells such as is done in cryopreservation and cryosurgery.
In this chapter the fundamental question of how does protein-DNA or protein-RNA interaction affect the structures and dynamics of DNA, RNA and protein is addressed. Models for calf-thymus DNA and transfer RNA interactions with human serum albumin (HSA), ribonuclease A (RNase A) and deoxyribonuclease I (DNase I) are presented here, using Fourier Transform Infrared (FTIR) spectroscopy in conjunction with UV-visible and CD spectroscopic methods. In the models considered, the binding sites, stability and structural aspects of protein-DNA and protein-RNA are discussed and the effects of protein interaction on the secondary structures of DNA and protein were determined.
The last 15 years have witnessed the development of modern infrared spectroscopy into a useful biodiagnostic tool for the analysis of cells, tissues, and body fluids. Dedicated technologies have evolved for rapidly discriminating between diverse microorganisms, testing single cells, and identifying various disease states in humans and animals. Particularly interesting applications arose by means of light microscopes coupled to infrared spectrometers. Infrared microscopes equipped with focal plane array detectors allow routinely the parallel collection of thousands of pixel spectra across microscopic areas of biological samples. This imaging technology can be used for automatic histological segmentation and imaging of tissue structures without dyes or molecular probes. Recent biomedical studies have proven that FT-IR imaging can be used to objectively differentiate benign from malignant histopathological structures in various tissues. Basically the same experimental set-up is also well suited to integrate the fundamental tasks of microbiological analysis, namely detection, enumeration, and differentiation of micro-organisms in one single apparatus. Due to its high brilliance, IR-synchrotron light coupled into high-quality FT-IR microscopes has been used for spectral mapping of single cells at a spatial resolution near the diffraction limit of mid-infrared light. The problem of extracting the characteristic information from the typically very complex, fingerprint-like infrared signatures of biological samples is generally addressed by applying bioinformatic techniques such as factor-, cluster-, linear discriminant analysis, and artificial neural networks together with so-called “feature extraction” algorithms. Examples are given on the characterization of micro-organisms, analysis of single eukaryotic cells, imaging of diseased human tissues, and disease recognition from body fluids that highlight the new possibilities of modern biomedical infrared spectroscopy.
This Chapter discusses the latest advances in the application of Near Infrared Spectroscopy (NIRS) in biomedical science.
Synchrotron infrared microscopy exploits the so-called brightness (or brilliance) advantage of synchrotron radiation. The main characteristics of such a source, coupled with an infrared microscope, are reviewed. Several important applications in the biomedical field are summarized including drug effects on cancer cells, growth factor signaling in single cells, compositional changes in microdamaged bone, infectious prion protein structure in nervous tissues, human substantia nigra in Parkinson's disease and β-amyloid deposition in Alzheimer's disease.