The research described in this thesis aims at exploiting recent important theoretical and experimental developments in protein folding towards developing advanced biosensors with improved/novel properties. Transforming this basic protein folding knowledge into engineering strategies for designing novel biosensors is an enormous challenge. Here we demonstrate the very first steps in that direction. Biosensors based on proteins that naturally toggle between two states (unfolded/folded) upon specific binding to a target molecule have been successfully used for real-time sensing. These devices behave as conformational switch sensors. Principles on how to engineer this type of conformational transducer onto any protein of interest have also been laid out. Our work takes this state of the art forward to design high-performance conformational rheostat sensors. The rationale is to develop sensors with expanded dynamic range and faster response time by using as conformational transducer the coupling of binding to the analyte and the gradual folding process of fast folding protein modules, and fluorescence as optical readout. As proof of concept, we investigate the pH sensing capabilities of engineered proteins based on two scaffolds: i) an anti-parallel coiled-coil, and ii) a tandem array of the small downhill folding domain BBL. Our results reveal that such pH sensors exhibit a linear response over at least 4 orders of magnitude in proton concentration. We also demonstrate that a pH sensor based on a conformational rheostat transducer can produce analog pH readouts at the single-molecule level together with ultrafast response (

Proteins are the work horses of the cell that perform the vast majority of functions essential for life. The mechanism by which proteins fold to their functional native state has been a subject of extensive research for more than 50 years now. Downhill folders are the class of proteins whose folding reaction is heterogeneous, non-cooperative, and happens without encountering a significant free energy barrier, resulting in ultrafast kinetics. The single ensemble of conformations of a global downhill folding protein moves gradually from highly disordered to the unique native structure when thermodynamic parameters that affect the protein's stability are changed (one-state folding). The gradual morphing of a one-state downhill folding protein structure in response to thermodynamic bias is referred to as a conformational rheostat. When such a conformational rheostat is coupled to binding an analyte, it can result in an ultrafast, broad band, and single-molecule analog biosensors. This thesis explores conformational rheostats as the mechanism behind the folding upon binding behavior of intrinsically disordered proteins and as broadband transducers towards engineering high-performance biosensors.The second chapter of this thesis describes a new methodology that we have developed to study the conformational landscape of intrinsically partially disordered proteins (IPDP). This methodology is inspired by the LEGO game, where the sequence of an IPDP is deconstructed into its local structural elements and their possible combinations based on the 3D structure the IPDP acquires upon binding its partners. The local structural elements are hence analogous to LEGO building blocks, and their combinations report on the interactions among them, like the complementary indentations of LEGO pieces. In particular, we chose the IPDP NCBD as model IPDP to develop the proof of concept for the method. Our results showed that even though the NCBD is highly flexible and apparently disordered, there are strong local signals and different sets of long-range transient interactions. These sets of interactions stabilize the overall fold and compete with one another hence resulting in a dynamic ensemble. The methodology developed in Chapter 2 is expected to be extremely useful in characterizing the incipient cooperativity of virtually any IPDP in their unbound form, a capability that is currently unavailable. Chapters 3 and 4 of this thesis deal with the design of a pH biosensor using the downhill folding protein gpW as a scaffold and unfolding coupled to ionization as a transducer. In chapter 3, a methodology for engineering conformational pH transducing into pH insensitive proteins using a histidine grafting approach was developed. The methodology was applied to the protein gpW to demonstrate an engineered, tunable broadband pH transducer based on the conformational rheostat mechanism. Chapter 4 explores general strategies for introducing fluorescence readouts capable of converting the gradual conformational changes of the rheostatic pH transducer into broadband fluorescence-based pH biosensors. Strategies that exploit the Förster Resonance Energy Transfer (FRET) and Photo Induced Electron Transfer (PET) mechanisms were explored as potential means to convert changes in conformation into suitable fluorescence signals were explored and characterized. We discovered that FRET signals using fluorophores in the visible (required for high-sensitivity biosensing) are insensitive to the localized conformational changes associated with conformational rheostats in native-like conditions. In contrast, the very short-range distance dependence of PET (

Fluorescent Analogs of Biomolecular Building Blocks focuses on the design of fluorescent probes for the four major families of macromolecular building blocks. Compiling the expertise of multiple authors, this book moves from introductory chapters to an exploration of the design, synthesis, and implementation of new fluorescent analogues of biomolecular building blocks, including examples of small-molecule fluorophores and sensors that are part of biomolecular assemblies.

The topics range from single molecule experiments in quantum optics and solid-state physics to analogous investigations in physical chemistry and biophysics.

The ribosome is a macromolecular machine that synthesizes proteins with a high degree of speed and accuracy. Our present understanding of its structure, function and dynamics is the result of six decades of research. This book collects over 40 articles based on the talks presented at the 2010 Ribosome Meeting, held in Orvieto, Italy, covering all facets of the structure and function of the ribosome. New high-resolution crystal structures of functional ribosome complexes and cryo-EM structures of translating ribosomes are presented, while partial reactions of translation are examined in structural and mechanistic detail, featuring translocation as a most dynamic process. Mechanisms of initiation, both in bacterial and eukaryotic systems, translation termination, and novel details of the functions of the respective factors are described. Structure and interactions of the nascent peptide within, and emerging from, the ribosomal peptide exit tunnel are addressed in several articles. Structural and single-molecule studies reveal a picture of the ribosome exhibiting the energy landscape of a processive Brownian machine. The collection provides up-to-date reviews which will serve as a source of essential information for years to come.

Protein folding and aggregation is the process by which newly synthesized proteins fold into the specific three-dimensional structures defining their biologically active states. It has always been a major focus of research in biochemistry and has often been seen as the unsolved second part of the genetic code. In the last 10 years we have witnessed a quantum leap in the research in this exciting area. Computational methods have improved to the extent of making possible to simulate the complete folding process of small proteins and the early stages of protein aggregation. Experimental methods h.

This book examines the physics, chemistry, and structure-property relationships of nanomaterials, as well as nanoscale electronics and photonics devices. It also discusses metal and semiconductor clusters, composites, thin films, and molecular engineering.