TIBS 17 - SEPTEMBER 1992 and the molecular basis of the functional heterogeneity of the muscarinic acetylcholine receptor.~ Relevant to neural signalling, he also pursued his work on the precursors of opioid peptides and corticotropin-releasing factor, guanine nucleotide-binding regulatory proteins (G proteins), and Na÷/K+-transportingATPase. For these outstanding achievements, he was awarded numerous honours and
awards including, most outstandingly, Person of Cultural Merits (1991), the Japan Academy prize (1985), the F. O. Schmitt Medal and Prize in Neuroscience (1990) and the Otto-WarburgMedaille (1987). He was also a foreign associate of the National Academy of Sciences USA since 1991 and a member of Leopoldina since 1990. Besides being a devoted scientist, Professor Numa
was a warm and friendly person by nature. We all mourn the loss of this valued colleague whose pioneering work in neuroscience and molecular biology will long be remembered. OSAMU HAYAISHI
Osaka Bioscience Institute, 6-2-4 Furuedai Suita, Osaka 565, Japan.
TECHNIUES DETERMINATION OF THE STRUCTURE of a biomolecule is often the most critical step towards reaching an understanding of its mechanism of action. The number of proteins of known amino acid sequence is over 40000, while detailed structural information is available for only several hundred. It is now possible to carry out site-directed mutagenesis and obtain mutant proteins for structure-function relationship studies. While functional studies on these proteins are often relatively easy, structural studies continue to be the rate-limiting step. Structural biologists are therefore constantly searching for new techniques or improvements of existing methods to increase the pace of protein structural determinations. The complete three-dimensional structure of a protein at high resolution can be determined by X-ray crystallography and a few closely related diffraction techniques. However, these techniques require the molecule to form a wellordered crystalline array, which is not possible for all proteins. A viable alternative to diffraction techniques is multi-dimensional nuclear magnetic resonance (NMR) spectroscopy. This technique has an advantage over crystallography in that structures of the proteins can be determined in solution. The major drawback of NMR spectroscopy, however, is that the interpretation of NMR spectra of large proteins is complex, so" its present
p. I. Haris and D. Chapman are at the Department of Protein and Molecular Biology, Royal Free Hospital School of Medicine, University of London, Rowland Hill Street, London, UK NW3 2PF.
328
Does Fourier-transform infrared spectroscopy provide useful information on protein structures?
Interest is growing in the application of Fourier-transform infrared (FIR) spectroscopy to the study of biomolecules in an aqueous environment. The increasing popularity of the technique is due to its ease of application to determine the secondary structure of peptides, proteins and also membrane proteins within their native lipid bilayer matrix. The ability to probe structural changes at a molecular level as a function of light, heat, pH or other external factors using difference spectroscopy is making a detailed understanding of the mechanism of action of different proteins possible. The use of isotopically labelled molecules helps in both the assignment of spectra and the attainment of greater specificity, and provides a new approach for the study of protein-protein interactions.
application is limited to small proteins (~15-20 kDa). Compared with the many structural studies of soluble proteins, the threedimensional structures of only three or four membrane proteins have been determined. This is because of the inability to produce high-quality threedimensional crystals of membrane proteins for X-ray diffraction studies. Membrane proteins have to be retained in a hydrophobic environment and experimentalists resort to producing detergent-protein crystals for such studies. As yet, no complete threedimensional structure exists for a membrane protein intact in its lipid bflayer matrix. Electron diffraction techniques
have been applied to some membrane proteins in their native lipid bilayer (e.g. bacteriorhodopsin ~) but so far the resolution obtained does not match what in principle can be achieved using the X-ray single crystal approach. NMR spectroscopy is severely restricted for the study of membrane proteins because of the non-isotropic motion of the proteins within the lipid bilayer matrix. A few studies have been made with some small membrane-associated polypeptides and proteins in membranemimetic environments, such as in micelles and organic solvents, but these studies do not necessarily provide an accurate picture of the arrangement of the protein within the lipid bilayer. © 1992,ElsevierSciencePublishers,(UK)
TIBS 17 - SEPTEMBER1992
The various limitations encountered with high-resolution structural studies of proteins have led to the development of alternative methods that provide different insights into protein structure without providing the total threedimensional structure. These methods include circular dichroism (CD) and vibrational spectroscopy [Raman and infrared OR)]. CD is a well-established technique for the analysis of the secondary structure of proteins in aqueous solution. This technique seems to be less reliable for the study of membrane proteins due to light-scattering problems associated with large membrane fragments. Raman spectroscopy is also a useful technique for the study of proteins in solution as well as in other environments. A disadvantage of Raman spectroscopy is that it is often difficult to obtain spectra of high signal-to-noise ratio because of interfering luminescence background. Even Fouriertransform Raman spectroscopy can give spectra of proteins which are dominated by that of the chromophore rather than that of the protein itself 2. Resonance Raman spectroscopy is a powerful method for the structural investigation of chromophores in biological molecules. A drawback of the technique is that it provides selective information on the chromophore without providing any significant insight into the structure of the protein or polypeptide. A particular feature of the new technique of Fourier-transform infrared spectroscopy (FTIR) for the study of proteins is that high-quality spectra can be obtained with relative ease with very small amounts of protein (1 mM) in a variety of environments such as aqueous solution, lipids, crystals and organic solvents. There are no problems associated with background fluorescence, light scattering or the size of the molecule. FTIR spectroscopy, unlike fluorescence and resonance Raman spectroscopy, is not limited to providing information on the chromophores, as it can monitor absorption from all bonds of the biomolecule. Furthermore, FTIR spectroscopy does not rely o n the use of additional probe molecules, as is required with some spectroscopic methods.
Basicprinciplesof IR spectroscopy Chemical bonds undergo various forms of vibrations such as stretching, twisting and rotating. The energy of most molecular vibrations corresponds
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Figure1 FTIR absorbance spectra showing th,e amide I and amide IIbands of two soluble proteins in aqueous solution obtained after subtraction of the background H20 absorption. (a) The absorbance spectrum of myoglobin (continuous line), a predominantly c(-helical protein. (b) The absorbance spectrum of concanavalin A (broken line), a predominantly ~-sheet containing protein.
to that of the IR region of electromagnetic spectrum. There are two main types of spectroscopic methods based on the vibration of the atoms of a molecule, namely IR and Raman spectroscopy. Raman spectroscopy is sensitive to vibrations that modulate bond polarizability. Vibrations that lead to changes in the dipole moment of a molecule can be detected and measured using IR spectroscopy. The possible number of modes of vibrations is 3N - 6 for a molecule consist!ng of N atoms (3N - 5 if the molecule is linear). Thus for a biomolecule such as a protein there are many vibrations which can result in a complex spectrum. Fortunately, however, many of the vibrations can be localized to specific bonds or groupings, such as the C=O and O-H groups. This has led to the concept of characteristic group frequencies. Typical group frequencies of interest to biochemists include C=O, -COOH, COO-, O-H and S-H. There are many vibrational modes that do not represent
a single type of bond oscillation but are strongly coupled to neighbouring bonds. For example, the IR spectrum of a protein is characterized by a set of absorption regions known as the amide modes.
Instrumentationand data processing IR spectroscopy is one of the earliest techniques to be applied to the study of protein structure. The potential of the technique was first demonstrated by the pioneering work of Elliott and Ambrose3 soon after the development of commercial dispersive IR instruments. The advent of computerized Fourier-transform instrumentation~ has made IR spectroscopy amenable to the study of biological systems. The underlying water absorption can be mathematically subtracted with great accuracy from an aqueous solution spectrum, making the study of hydrated biological systems almost routine. Figure 1 shows the absorbance spectra of two soluble proteins after the digital subtraction of
329
TIBS 17 - SEPTEMBER 1992
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awards including, most outstandingly, Person of Cultural Merits (1991), the Japan Academy prize (1985), the F. O. Schmitt Medal and Prize in Neuroscience (1990) and the Otto-WarburgMedaille (1987). He was also a foreign associate of the National Academy of Sciences USA since 1991 and a member of Leopoldina since 1990. Besides being a devoted scientist, Professor Numa
was a warm and friendly person by nature. We all mourn the loss of this valued colleague whose pioneering work in neuroscience and molecular biology will long be remembered. OSAMU HAYAISHI
Osaka Bioscience Institute, 6-2-4 Furuedai Suita, Osaka 565, Japan.
TECHNIUES DETERMINATION OF THE STRUCTURE of a biomolecule is often the most critical step towards reaching an understanding of its mechanism of action. The number of proteins of known amino acid sequence is over 40000, while detailed structural information is available for only several hundred. It is now possible to carry out site-directed mutagenesis and obtain mutant proteins for structure-function relationship studies. While functional studies on these proteins are often relatively easy, structural studies continue to be the rate-limiting step. Structural biologists are therefore constantly searching for new techniques or improvements of existing methods to increase the pace of protein structural determinations. The complete three-dimensional structure of a protein at high resolution can be determined by X-ray crystallography and a few closely related diffraction techniques. However, these techniques require the molecule to form a wellordered crystalline array, which is not possible for all proteins. A viable alternative to diffraction techniques is multi-dimensional nuclear magnetic resonance (NMR) spectroscopy. This technique has an advantage over crystallography in that structures of the proteins can be determined in solution. The major drawback of NMR spectroscopy, however, is that the interpretation of NMR spectra of large proteins is complex, so" its present
p. I. Haris and D. Chapman are at the Department of Protein and Molecular Biology, Royal Free Hospital School of Medicine, University of London, Rowland Hill Street, London, UK NW3 2PF.
328
Does Fourier-transform infrared spectroscopy provide useful information on protein structures?
Interest is growing in the application of Fourier-transform infrared (FIR) spectroscopy to the study of biomolecules in an aqueous environment. The increasing popularity of the technique is due to its ease of application to determine the secondary structure of peptides, proteins and also membrane proteins within their native lipid bilayer matrix. The ability to probe structural changes at a molecular level as a function of light, heat, pH or other external factors using difference spectroscopy is making a detailed understanding of the mechanism of action of different proteins possible. The use of isotopically labelled molecules helps in both the assignment of spectra and the attainment of greater specificity, and provides a new approach for the study of protein-protein interactions.
application is limited to small proteins (~15-20 kDa). Compared with the many structural studies of soluble proteins, the threedimensional structures of only three or four membrane proteins have been determined. This is because of the inability to produce high-quality threedimensional crystals of membrane proteins for X-ray diffraction studies. Membrane proteins have to be retained in a hydrophobic environment and experimentalists resort to producing detergent-protein crystals for such studies. As yet, no complete threedimensional structure exists for a membrane protein intact in its lipid bflayer matrix. Electron diffraction techniques
have been applied to some membrane proteins in their native lipid bilayer (e.g. bacteriorhodopsin ~) but so far the resolution obtained does not match what in principle can be achieved using the X-ray single crystal approach. NMR spectroscopy is severely restricted for the study of membrane proteins because of the non-isotropic motion of the proteins within the lipid bilayer matrix. A few studies have been made with some small membrane-associated polypeptides and proteins in membranemimetic environments, such as in micelles and organic solvents, but these studies do not necessarily provide an accurate picture of the arrangement of the protein within the lipid bilayer. © 1992,ElsevierSciencePublishers,(UK)
TIBS 17 - SEPTEMBER1992
The various limitations encountered with high-resolution structural studies of proteins have led to the development of alternative methods that provide different insights into protein structure without providing the total threedimensional structure. These methods include circular dichroism (CD) and vibrational spectroscopy [Raman and infrared OR)]. CD is a well-established technique for the analysis of the secondary structure of proteins in aqueous solution. This technique seems to be less reliable for the study of membrane proteins due to light-scattering problems associated with large membrane fragments. Raman spectroscopy is also a useful technique for the study of proteins in solution as well as in other environments. A disadvantage of Raman spectroscopy is that it is often difficult to obtain spectra of high signal-to-noise ratio because of interfering luminescence background. Even Fouriertransform Raman spectroscopy can give spectra of proteins which are dominated by that of the chromophore rather than that of the protein itself 2. Resonance Raman spectroscopy is a powerful method for the structural investigation of chromophores in biological molecules. A drawback of the technique is that it provides selective information on the chromophore without providing any significant insight into the structure of the protein or polypeptide. A particular feature of the new technique of Fourier-transform infrared spectroscopy (FTIR) for the study of proteins is that high-quality spectra can be obtained with relative ease with very small amounts of protein (1 mM) in a variety of environments such as aqueous solution, lipids, crystals and organic solvents. There are no problems associated with background fluorescence, light scattering or the size of the molecule. FTIR spectroscopy, unlike fluorescence and resonance Raman spectroscopy, is not limited to providing information on the chromophores, as it can monitor absorption from all bonds of the biomolecule. Furthermore, FTIR spectroscopy does not rely o n the use of additional probe molecules, as is required with some spectroscopic methods.
Basicprinciplesof IR spectroscopy Chemical bonds undergo various forms of vibrations such as stretching, twisting and rotating. The energy of most molecular vibrations corresponds
1654;r~ A~"1635 Amide I Myoglobin
Jl Amide I I Concanavalin A
0c-Helix
I 3-Sheet
Amide II
1548~i538
o=
i
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I ~
1
1800
I
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i
i
i
I
1700 '
I
1600
ni1
~
I
I1
1500
l
1
I
00
14
Wavenumber (cm -1)
Figure1 FTIR absorbance spectra showing th,e amide I and amide IIbands of two soluble proteins in aqueous solution obtained after subtraction of the background H20 absorption. (a) The absorbance spectrum of myoglobin (continuous line), a predominantly c(-helical protein. (b) The absorbance spectrum of concanavalin A (broken line), a predominantly ~-sheet containing protein.
to that of the IR region of electromagnetic spectrum. There are two main types of spectroscopic methods based on the vibration of the atoms of a molecule, namely IR and Raman spectroscopy. Raman spectroscopy is sensitive to vibrations that modulate bond polarizability. Vibrations that lead to changes in the dipole moment of a molecule can be detected and measured using IR spectroscopy. The possible number of modes of vibrations is 3N - 6 for a molecule consist!ng of N atoms (3N - 5 if the molecule is linear). Thus for a biomolecule such as a protein there are many vibrations which can result in a complex spectrum. Fortunately, however, many of the vibrations can be localized to specific bonds or groupings, such as the C=O and O-H groups. This has led to the concept of characteristic group frequencies. Typical group frequencies of interest to biochemists include C=O, -COOH, COO-, O-H and S-H. There are many vibrational modes that do not represent
a single type of bond oscillation but are strongly coupled to neighbouring bonds. For example, the IR spectrum of a protein is characterized by a set of absorption regions known as the amide modes.
Instrumentationand data processing IR spectroscopy is one of the earliest techniques to be applied to the study of protein structure. The potential of the technique was first demonstrated by the pioneering work of Elliott and Ambrose3 soon after the development of commercial dispersive IR instruments. The advent of computerized Fourier-transform instrumentation~ has made IR spectroscopy amenable to the study of biological systems. The underlying water absorption can be mathematically subtracted with great accuracy from an aqueous solution spectrum, making the study of hydrated biological systems almost routine. Figure 1 shows the absorbance spectra of two soluble proteins after the digital subtraction of
329
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