Book Review

How a protein molecule folds to its native, functional state from the literally billions of alternative possibiliti es has fascinated biologists for years. Not only will an understanding of folding be of enormous intellectua l satisfaction, providing new insights into the origins of protein structure and stability, but it will also open the door to new experiments, including designing proteins de novo and developing therapies for folding diseases. Protein folding is a complex event (see Brockwell et al. (2000) and Dinner et al. (2000) for reviews) and, as a consequence, advances in this field have often relied on new experimenta l approaches and theoretica l innovations. The author sets out this book as an introduction to rapid kinetic methodologies and their analysis to monitor protein chemical reactions with an emphasis on protein folding. It focuses on the results of theauthor and his co-workers (NoÈlting et al. 1995, 1997, NoÈlting 1996, 1999), references to work from other groups can be found in the thorough bibliography. The book is aimed at students of mathematics , biophysics , biotechnology, biochemistry and medicine, who are interested in practical methods of measuring kinetic processes and fitting and modelling their data. The book opens with three short chapters that discuss protein structure and the physica l forces that stabilize the native state, briefly defining the principles of the interactions that govern the final functional structure. This sets the scene for the following chapters, but readers wishing to gain more insights into protein structure and stability will need to consult oneor moreof the excellentalternative texts thatare dedicated to these subjects . The fourth chapter focuses on the derivation of kinetic rate constants and kinetic solutions of various reaction mechanisms . This chapter will be a useful reference for anyone interested in kinetic processes. Here, the author provides a comprehensiv e derivation of the rate constants for different mechanisms that may describe a folding protein, including simple two-state transitions (folding without populated intermediates ), three-state transitions (folding via an intermediate), and a host of more complex kinetic scenarios including off-pathway intermediates, branched pathways, and protein multimerization. The analytical solution for mechanisms with up to four separate thermodynamic states are listed, as are the changes in populations of the different components as the reaction proceeds to completion. Disappointingly, the solutions of the amplitudes for each rate constant are not provided. Nevertheless , these schemes, summarized in a twopage table, provide a very useful source of information for those wishing to understand kinetic analyses or simply to pick out suitable equations with which to fit their results. The book fulfils its objective in describing the methods now available to measure high-resolution kinetics in the following two chapters . These will be of interest to a wide audience, including biochemists and physicists . The author outlines the principles behind the techniques of ultra-rapid mixing, temperature jump, optical triggers , acoustic relaxation, pressurejump, dielectric relaxation and NMR broadening. For each method, the requirements of the model to be studied and schematic diagrams of the equipment are provided. Unfortunately, the author does not show examples of real experimental data obtained using these methods (although useful references are cited). The text also introduces other methods used for monitoring kinetic processes on much slower timescales and builds a picture of how rates of reaction may be measured from nanosecond to months temporal resolution (including NMR, time resolved CD, and isotope labelling). Phi-value analysis is a powerful method that can be used to characterize the barriers in protein folding (i.e. the high energy transition states) in a residue-specific manner. The approach was first described and implemented by Fersht (1999). Simply put, the method relies on making a host of site-directed mutants, determining the energetic cost of the sequence change on the folding and unfolding rate constants , and using this to infer structural information. NoÈlting briefly discusses molecula r biological techniques in his book, and mentions problems that students might encounter in expressing proteins. Much more thorough descriptions of these topics can be found elsewhere, although these brief sections at least alert students about potential pitfalls and their possible avoidance. The reader will find a clear explanation of how to measure kinetic rate constants , derive and fit equilibr ium denaturation curves and measure, calculate and interpret phi values for proteins that fold with mechanisms of different complexity. The book culminates in a chapter which describes the folding of barstar at the level of individual amino acids, on a lsec to sec timescale. The reader, at last, can see real experimenta l data and howthe methods and analyses can be applied to determine the kinetic folding mechanism of this small a /b protein. Does this book meet its aims? The answer is at least partly so. It will appeal to those working in the folding field, and particularly those interested in instrumentation and kinetic analyses. At £57.50, the book is not cheap, but will be a good buy for researchers fulfilling these criteria. Researchers with less methodological or mathematical interests might be better to look elsewhere, and those interested in membrane proteins will have to work out for themselves howto apply the methods and analyses to these fascinating, but more difficult systems. Even for these researchers, however, the book will be a useful source of information that is well worth a place on the library shelf. Molecular Membrane Biology, 2000, 17, 185± 186