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Preface
Advancement in the field of structural biology is critical to the advancement of agricultural, biomedical, and pharmaceutical science and technology. In this regard, determination of protein structure and also the correlation between structure and function form the core issues of modern molecular biology. The developments of the past decade in the increased application of IR spectroscopy have been very helpful for the analysis of structure and function relationship of peptides and proteins. However, IR spectroscopy is still not a commonly used method for this purpose, and remains an optional method for experts only. Issues related to protein IR analysis range from instrumentation and sampling methods to theoretical and experimental band assign ments and derivations. To address some of these issues, an international symposium on Infrared Analysis of Peptides and Proteins was organized during the 216 Annual Meeting of the American Chemical Society (ACS), August 23-27, 1998, at Boston, Massachusetts. th
Several spectroscopic techniques are available for studying protein structures, including circular dichroism, U V absorption and fluorescence, Raman, NMR, and IR spectroscopy. However, each of these techniques has its limitations. For example, U V absorption and fluorescence spectroscopic techniques are generally confined to topography of aromatic amino acids. Among the techniques for secondary structure, N M R and Raman spectroscopy need unusually high concentration of proteins, and N M R analysis is generally limited to small proteins, so far. Circular dichroism analysis is limited to clear protein solutions (as opposed to membrane proteins) due to the prob lem with light scattering. IR spectroscopy is perhaps the most suited technique for protein analysis because of its sensitivity, versatility, and responsiveness to structural changes. IR spectroscopy is being widely used as a probe of protein molecular structure at the secondary level and has potential for tertiary structural level analysis. This method has been recognized as a tool for studying conformation in polypeptides and proteins since 1950 when Elliot and Ambrose found that polypeptides known to be in the ahelix and β-sheet forms, displayed absorbance of the C=0 stretching and N - H defor mation modes characteristically at different frequencies. In an attempt to find the origin of characteristic peptide vibrations, Miyazawa and co-workers in the 1950s determined the chemical groups that give rise to the charac teristic amide vibrations using peptide analogues. The amide I, Π, and ΠΙ vibrational modes were identified to be localized within the peptide CONH group, and became the focus of most attempts to correlate polypeptide and protein secondary structure with IR spectral properties, with the amide I mode being used the most because of its relative vii Singh; Infrared Analysis of Peptides and Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1999.
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strength. In the 1960s and 1970s, experimental studies by Timasheff, Susi, and others and theoretical normal mode calculations by Shimanouchi, Krimm, and others solid ified our understanding of IR spectra-structure correlations of amide bands of poly peptides and proteins. These studies showed that amide vibrational frequencies assoc iated with different conformations in model systems were roughly transferable to proteins, in general, for the α-helix, β-sheet, and unordered conformations. The infor mation from these studies and theoretical calculations relating spectral characteristics to protein conformations were used to predict the structural composition of globular proteins. The advent of modern Fourier transform infrared (FTIR) spectrometers has led to significant advances in IR spectroscopy, particularly in protein conformational analy sis. The digital nature of FTIR spectroscopic data, combined with high signal-to-noise ratio, also allows the use of "resolution-enhancement" techniques. Derivative spectros copy and Fourier self-deconvolution (FSD) enhance the spectroscopist's ability to visualize component peaks in multicomponent, overlapping bands such as those arising from conformation-sensitive modes in protein spectra. These approaches along with curve-fitting process, promoted by Chapman, Mantsch, Susi, and others, have been exploited extensively in the 1980s and 1990s to obtain reasonable estimates of secon dary structure contents of peptides and proteins. Current FTIR methods of determ ination of protein conformation involve analysis of the amide I and amide III bands employing curve-fitting methods for the component peaks disclosed by resolution enhancement techniques. In addition, statistical methods of multifactor regression analysis are now being employed to extract secondary structure contents from amide I, Π, and III spectra. Within the past few years, there has been tremendous development in several areas that are relevant to the effective use of IR spectroscopy to analyze proteins. These include: availability of modern computers, interfaces of FTIR spectrometers to multiple variables (temperature, pressure, and titrators), different sampling techniques; optical fiber probes, chemometric approaches, fast data collection methods for timeresolved analysis, and polarization methods. Additionally, great advances have oc curred in the biotechnology of sample preparations such as isotopic labeling of proteins, which allows fine-tuning of protein signals and assignments. While theoret ical calculations of IR band positions are still less than adequate for use by themselves, advancement in the field at least has been complementary to experimental results. It is imperative that these advances (1) continue and be developed synergistically, and (2) be communicated to user scientists who are in dire need of structural information about proteins. The symposium and the resulting symposium volume monograph are steps toward these goals. Although this book by no means represents all the work in the field that has been carried out or is in progress, it has captured the essence of the basic foundations as well as recent advances in the application of IR spectroscopy for the analysis of peptides and proteins. I feel that there is a need for a volume like this to apprise researchers in the area of protein structure and functions of the possibilities that are currently available with IR spectroscopy. This observation was in part supported by the fact that the symposium itself was very well attended. Notably, many attendees were from the industrial research groups.
viii Singh; Infrared Analysis of Peptides and Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1999.
The volume has chapters ranging from the theoretical basis of the protein IR signals to the application of IR spectroscopy to resolve biological events at nanosecond scale. The chapters are written to include basic introductions of the subject matter, and have extensive examples of applications with graphics and references. The volume could make a reference book for an advanced biological spectroscopy course at graduate level.
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Acknowledgments Organizing a symposium of this nature, and the preparation of a symposium volume, requires coordination of efforts by several individuals. I am very fortunate to have the assistance of very capable people like Henry Blount and Ed Yeung, who initially encouraged me with this project, and provided guidance on the details of the symposium program. J. Michael Ramsey was instrumental in finalizing the program, and along with Michèle Buchanan, in arranging for support from the ACS Division of Analytical Chemistry. The ACS Petroleum Fund and the National Cancer Institute of the National Institutes of Health (R13CA79103) provided critically needed funds to support symposium speakers, and the University of Massachusetts at Dartmouth provided the administrative support. Preparation of the book was coordinated by Anne Wilson and Kelly Dennis of the ACS Books Department. Their patience and assistance in maintaining the timeline for getting the manuscripts processed were invaluable to the completion of this volume. Finally, I am very grateftil to the contributors of the volume, who willingly accepted the deadlines and patiently made changes suggested by a dedicated group of reviewers of the chapter manuscripts. B A L R A M SINGH
Department of Chemistry and Biochemistry and Center for Marine Science and Technology University of Massachusetts at Dartmouth Dartmouth, MA 02747
ix Singh; Infrared Analysis of Peptides and Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1999.
