Structural characterization of membrane proteins and peptides by FTIR and ATR-FTIR spectroscopy (original) (raw)
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Structural analysis of proteins by isotope-edited FTIR spectroscopy
Spectroscopy, 2010
Structure determination of multidomain proteins or protein–membrane complexes is one of the most challenging tasks in modern structural biology. High-resolution techniques, like NMR or X-ray crystallography, are limited to molecules of moderate size or those that can be crystallized easily. Both methods encounter serious technical obstacles in structural analysis of protein–membrane systems. This work describes an emerging biophysical technique that combines segmental isotope labeling of proteins with Fourier transform infrared (FTIR) spectroscopy, which provides site-specific structural information on proteins and allows structural characterization of protein–membrane complexes. Labeling of a segment of the protein with13C results in infrared spectral resolution of the labeled and unlabeled parts and thus allows identification of structural changes in specific domains/segments of the protein that accompany functional transitions. Segmental isotope labeling also allows determination...
Biomedical Spectroscopy and Imaging, 2016
Membrane proteins facilitate some of the most important cellular processes including energy conversion, ion transport and signal transduction. While conventional infrared absorption provides information about membrane protein secondary structure, a major challenge is to develop a dynamic picture of the functioning of membrane proteins at the molecular level. The introduction of FTIR difference spectroscopy around 1980 to study structural changes in membrane proteins along with a number of associated techniques including protein isotope labeling, site-directed mutagenesis, polarization dichroism, attenuated total reflection and time-resolved spectroscopy have led to significant progress towards this goal. It is now possible to routinely detect conformational changes of individual amino acid residues, backbone peptides, binding ligands, chromophores and even internal water molecules under physiological conditions with time-resolution down to nanoseconds. The advent of ultrafast pulsed-IR lasers has pushed this time-resolution down to femtoseconds. The early development of FTIR difference spectroscopy as applied to membrane proteins with special focus on bacteriorhodopsin is reviewed from a personal perspective.
FTIR spectroscopic characterization of protein structure in aqueous and non-aqueous media
Journal of Molecular Catalysis B: Enzymatic, 1999
With increasing use of proteins in many different applications, ranging from phramaceuticals to biosensors and biomaterials, there has emerged a need for protein structural characterisation in diverse environments. In many cases it is not sufficient to just have the three-dimensional structure of a protein in H O or in the crystalline state. Often information on the 2 structural properties of a protein is required in the presence of organic solvents, detergent micelles, phospholipid membranes Ž. and so on. Fourier transform infrared spectroscopy FTIR has been identified as one of the few techniques that can be applied for structural characterisation of proteins in such environments. Here we discuss how this technique is being used to obtain information on protein structure and stability in both aqueous and non-aqueous media. Examples are drawn from our studies of water soluble proteins and membrane proteins.
The Use and Misuse of FTIR Spectroscopy in the Determination of Protein Structure
Critical Reviews in Biochemistry and Molecular Biology, 1995
Fourier transform infrartd (FTIR) spectroscopy is an established tool for the structural characterization of proteins. However, many potential pitfalls exist for the unwary investigator. In this review we critically assess the application of FIlR spectroscopy to the determination of protein structure by (1) outlining the principles underlying protein secondary structure determination by FZZR spectroscopy. (2) highhghting the situations in which FZZR spectroscopy should be considered the technique of choice, (3) discussing the manner in which experiments should be conducted to derive as much physiologically relevant information as possible, and (4) outlining current methods for the determination of secondary structure from infrared spectm of proteins,
Rapid Characterization of Peptide Secondary Structure by FT-Ir Spectroscopy
Rev. Roum. Chim, 2011
Use of peptides and proteins in many different applications requires a more detailed structural characterization of these in different environments. Information on structural properties of peptides and proteins in various solvents are also given by Fourier transform infrared (FT-IR) spectroscopy. Here, we investigate the amide I, II, and III bands of peptides using both FT-IR spectra and their second derivatives. This paper reveals a large body of information obtained by FT-IR technique that can be used to understand the structure and stability of some peptides and proteins in different environments. Circular dichroism (CD) spectroscopy support data obtained within FT-IR experiments.
Qualification of FTIR spectroscopic method for protein secondary structural analysis
Journal of Pharmaceutical Sciences, 2011
Fourier transform infrared (FTIR) spectroscopy is widely used to study protein secondary structure both in solution and in the solid state. The FTIR spectroscopic method has also been employed as a characterization method by the biopharmaceutical industry to determine the higher order structure of protein therapeutics, and to determine if any changes in protein conformation have occurred as a result of changes to process, formulation, manufacture, and storage conditions. The results of these studies are often included in regulatory filings; when comparability is assessed, the comparison is often qualitative. To demonstrate that the method can be quantitative, and is suitable for these intended purposes, the precision and sensitivity of the FTIR method were evaluated. The results show that FTIR spectroscopic analysis is reproducible with suitable method precision, that is, spectral similarity of replicate measurements is greater than 90%. The method can detect secondary structural changes caused by pH and denaturant. The sensitivity of the method in detecting structural changes depends on the extent of the changes and their impact on the resulting spectral similarity and characteristic FTIR bands. The results of these assessments are described in this paper.
Journal of Physical Chemistry B, 2008
The amide I vibrational mode, primarily associated with peptide-bond carbonyl stretches, has long been used to probe the structures and dynamics of peptides and proteins by infrared (IR) spectroscopy. A number of ab initio-based amide I vibrational frequency maps have been developed for calculating IR line shapes. In this paper a new empirical amide I vibrational frequency map is developed. To evaluate its performance, we applied this map to a system of isotope-edited CD3-ζ membrane peptide bundles in aqueous solution. The calculated 2D-IR diagonal linewidths vary from residue to residue and show an asymmetric pattern as a function of position in the membrane. The theoretical results are in fair agreement with experiments on the same system. Through analysis of the computed frequency time-correlation functions, it is found that the 2D-IR diagonal widths are dominated by contributions from the inhomogeneous frequency distributions, from which it follows that these widths are a good probe of the extent of local structural fluctuations. Thus the asymmetric pattern of linewidths follows from the asymmetric structure of the bundle in the membrane.
Recent applications of ATR FTIR spectroscopy and imaging to proteins
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2013
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 crystallization and for highthroughput studies, highlighting the future potential of the technology within the field of protein structural studies and beyond.
Biopolymers, 2004
As more and more high-resolution structures of proteins become available, the new challenge is the understanding of these small conformational changes that are responsible for protein activity. Specialized difference Fourier transform infrared (FTIR) techniques allow the recording of side-chain modifications or minute secondary structure changes. Yet, large domain movements remain usually unnoticed. FTIR spectroscopy provides a unique opportunity to record 1 H/ 2 H exchange kinetics at the level of the amide proton. This approach is extremely sensitive to tertiary structure changes and yields quantitative data on domain/domain interactions. An experimental setup designed for attenuated total reflection and a specific approach for the analysis of the results is described. The study of one membrane protein, the gastric H ϩ ,K ϩ -ATPase, demonstrates the usefulness of 1 H/ 2 H exchange kinetics for the understanding of the molecular movement related to the catalytic activity.
Evaluation of the Protein Secondary Structures Using Fourier Transform Infrared Spectroscopy
gazi university journal of science, 2014
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. Fourier transform infrared spectrometry is well known as a powerful tool for determination of secondary structures of proteins. This paper presents the most recent experimental results obtained in our laboratory for this kind of analysis. A PeakFit operation was performed and some observations were made. Key words: FT-IR analysis, protein secondary structure.