Proteins are in charge of virtually every biological process that occurs in modern cells. In isolation or in complexes they often perform their functions through structural changes in their intra- or intermolecular bonding. Understanding the inner workings of these biological machines remains one of the major frontiers in biology. Many of these "bionanomachines" use mechanical forces in different cell processes ranging from replication, transcription, translation, and protein degradation to organelle transport, cell adhesion, and cell crawling. Several manipulation techniques can now be employed to investigate these mechanical processes at the single-molecule level, ttis review will start by introducing both protein machines and single-molecule manipulation techniques (Sect. 8.2).
Atomic force microscopy (AFM. Also used through the text as an acronym for atomic force microscope) in its single-molecule force spectroscopy mode (for simplicity, referred to as SMFS here; see Sect. 8.3) is one of the nanomanipulation techniques most extensively used for the study of the mechanical properties of proteins, particularly to examine their response to stretching (i.e., molecular elasticity and mechanical stability) and to unbinding. Several reviews have recently been dedicated to this topic, focusing on the analysis of intramolecular (Carrion-Vazquez et al. 2000; Isralewitz et al. 2001; Best et al. 2003a; Zhuang and Rief 2003; Bustamante et al. 2004; Rounsevell et al. 2004; Samori et al. 2005) or intermolecular interactions (Hinterdorfer 2002; Weisel et al. 2003), or both (Zlatanova et al. 2000; Merkel 2001).
tte aim of this review is to provide the nonspecialist with a quick guide so that she/he can become familiar with the central themes of this new and exciting field, tte main concepts, ideas, achievements, biological implications, and limitations will be discussed. Particular emphasis will be placed on the analysis of the intramolecular interactions of proteins (given that they have been more extensively characterized) and we will also highlight the advantages of combining this technique with protein engineering and molecular dynamics in order to elucidate the molecular determinants underlying the mechanical stability of proteins.
Springer Series in Biophysics J.L.R. Arrondo and A. Alonso Advanced Techniques in Biophysics © Springer-Verlag Berlin Heidelberg 2006
Comprehensive descriptions that provide the experimental details behind this new methodology can be found elsewhere (Carrion-Vazquez et al. 2000; Rounsevell et al. 2004). For an in-depth discussion on the role of mechanical forces in biochemistry, as well as for a review of the closely related field of motor proteins, see the works ofHoward (2001) and Bustamante et al. (2004).
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