ABSTRACT

Single- Molecule AFM ........................................................................50 2.2.3 Constructing Polyprotein to Pinpoint the Nanomechanics

of Protein Domains of Interest ........................................................... 52 2.2.4 How Applied Force Unfolds a Protein ................................................ 53 2.2.5 Mechanical Stability: A Kinetic, rather than

Thermodynamic, Stability ..................................................................54 2.2.6 Constructing the Free Energy Landscape for Protein

Unfolding from Single-Molecule AFM Experiments ........................ 55 2.2.7 Structural Features of Proteins Obtained

from Force-Extension Spectrum ........................................................ 58 2.2.8 Unfolding Force Depends on Pulling Direction: The Importance

of Local Structure to the Mechanical Stability of Proteins ................ 59 2.3 Tuning the Properties of Proteins: Tailoring Protein Mechanics

for Use with Single-Molecule AFM ...............................................................60 2.3.1 Present Modi¡cations and Their Application

with Single- Molecule AFM ...............................................................60 2.3.2 Introducing Point Mutations into the Native Protein Backbone:

Nanomechanical Effects of Single-Point Mutations ........................... 61 2.3.3 Recombination of Structural Fragments or Structural Grafting ........64 2.3.4 Intramolecular Disul¡de Bonding and Loop Insertion ...................... 67 2.3.5 Ligand Binding Modi¡es the Unfolding Energy Landscape ..............68 2.3.6 Investigating the Nanomechanics of a Novel Protein Fold: Top7 ....... 70 2.3.7 Environmental/Solvent Tuning of Mechanical Stability .................... 71 2.3.8 Predicting Mechanical Properties Using Modeling Approaches ....... 73

Investigating the nanomechanics of proteins, moving toward proteins that have been speci¡cally designed in order to exhibit certain characteristics, is an everevolving application of atomic force microscopy (AFM)-based single-molecule force spectroscopy. In little more than ten years, the ¡eld of protein mechanics has gone from simply observing the properties of naturally occurring proteins to charting the manner in which proteins with completely novel folds and uncharted functionality unfold when exposed to a denaturing force. This chapter seeks to explore the trajectory of single-molecule force spectroscopy, focusing on the application toward proteins with novel, engineered characteristics. To do so, this chapter is divided into four parts. Section 2.1 acts as a brief introduction to proteins, introducing structural/functional relationships that will form the backbone of the remainder of the chapter, as well as discussing proteins as a single-molecule AFM model system and the future bearing in which the ¡eld is directed. Section  2.2 discusses the application of single-molecule AFM in studying proteins, including how critical mechanical information is garnered. The application of these techniques to proteins that are tailored to display certain characteristics is discussed in Section 2.3, where an overview of presently employed protein modi¡cations, and the nanomechanical properties they display, is discussed. Section 2.4 deals with the future prospects of the ¡eld: The evolution and challenges to date, and how future innovations could lead to far reaching advances in ¡elds such as nanobiotechnology.