Single‐Molecule Spectroscopy of Unfolded Proteins

Abstract

13Single -molecule spectroscopy has developed into a versatile method to probe distances, distance distributions, and dynamics of unfolded proteins. Singlemolecule F ö rster resonance energy transfer has been used most extensively to study long -range intramolecular distance distributions and dynamics of unfolded proteins from timescales of nanoseconds to seconds . The methods developed in this context will also be helpful for studying the behavior of intrinsically disordered proteins. INTRODUCTIONThe folding of a protein is the fi rst part of its function: it has to fi nd its native three -dimensional structure -encoded in the amino acid sequence -to be able to act as an enzyme, a signal transducer, a membrane channel, or a stabilizing element of a cell, to name but a few of the many roles that proteins can take. Contrary to widespread belief, folding is not a unique, singular event in the life of a protein. Many proteins are marginally stable and will fold and unfold many times during their functional life. As described in the other chapters of ABSTRACT this book, in many cases proteins even fold only in the presence of stabilizing ligands or binding partners, illustrating the close coupling of folding and function. Correspondingly, the unfolded or denatured state is of central relevance to understanding the protein -folding reaction, especially in the context of intrinsically disordered proteins (IDPs) (24, 102) . SINGLE -MOLECULE SPECTROSCOPYIn the past decade, single -molecule methods have increasingly been employed to study protein folding. The two main methods used are force -probe techniques and F ö rster resonance energy transfer (FRET). Experiments using atomic force microscopy and laser tweezers have provided a lot of previously inaccessible information on the mechanical stability and folding of proteins, and the behavior of unfolded polypeptides under force, and the reader is referred to a number of recent reviews on this topic (7,11,27,28,111) . Here, we focus on the investigation of protein folding and especially unfolded proteins using single -molecule FRET ( Fig. 13.1 ). First demonstrated about 10 years ago (35) , single -molecule FRET has since become an important approach to study intramolecular distances and dynamics in biomolecules (106) , including protein folding (37, 62, 76, 86 -88) . FRETThe fi rst quantitative test of F ö rster ' s theory (29, 103) and the crucial experiment that put FRET on the map of biochemistry was published by Stryer and Haugland in 1967 (97) . They attached dansyl and naphthyl groups to the 372 INSTRUMENTAL ANALYSIS OF INTRINSICALLY DISORDERED PROTEINS

A novel amphiphilic thiosemicarbazone derivative for binding and selective sensing of human serum albumin

“…Design and development of new fluorescent probes for the study of biomolecules is the foundation of many biological processes . Fluorescence sensors are highly desirable because of their high sensitivity to fluorescence spectroscopy and light‐up probes, whose emission intensity increases when associated with biomolecules, and are useful markers in genomics and proteomics since binding events to the host molecule can result in the appearance of an intense fluorescence emission of the probes . Light‐up probes are mainly used for quantification of biomacromolecules and as stains in gel electrophoresis such as 1‐anilino‐8‐naphthalene sulfonate (1,8‐ANS), Nile Red and the SYPRO dye family .…”