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 (

In order to execute their biological activities, most proteins fold into their unique, three-dimensional structure. The discovery of intrinsically disordered proteins (IDPs) about two decades ago, which are now widely found in eukaryotes, has since challenged the structure-function paradigm. IDPs, which in isolation exist as broad, non-random, conformational ensembles of interconverting states, are centrally involved in many biological processes. The key to their functioning is the ability to fold when bound to ligand partner(s), thus operating as morphing proteins. Despite booming interest in morphing behavior, investigating their structural transitions and mechanism remains extremely difficult because of their distinct characteristics. Previously, we observed a close connection between intrinsically partially disordered proteins (IPDPS) and gradual (un)folding transitions of downhill folders, leading to the hypothesis that many IPDPs work as a conformational rheostat. The scope of this dissertation is to investigate the biological and technological implications of gradual conformational transitions. We first demonstrate the design principles of protein-based scaffolds by utilizing gradual (un)folding coupled to binding for developing rheostatic conformational transducers using computational modeling and experiments. Our engineered transducers showcase>6 orders of magnitude change in analyte concentration (broadband sensitivity) and have practical advantages over extant ones, which conventionally operate as conformational switches. Next, inspired by the LEGO toy, we devised a novel modular approach to dissect the folding cooperativity and the energetic contributions of native interactions in defining the conformational ensemble and binding properties of IPDPs. Using an integrated strategy of computation and experiments, we perform an ensemble-based conformational analysis and find that the approach provides an exciting new tool for analyzing morphing transitions that should generally apply to any IPDP, thereby addressing a fundamental gap in the field. One particularly interesting IPDP is NCBD that binds to multiple structurally diverse ligand partners and recruits the basal transcription machinery. We then explore the concept of NCBD functioning as a conformational rheostat, which allows its promiscuous binding. Finally, using extensive all-atom Molecular Dynamics simulations of NCBD and its biological partners in their free and bound forms, we decipher the hidden conformational biases in the dynamics of the heterogeneous ensemble of NCBD, undergoing gradual morphing transitions hinting at a working conformational rheostat in transcription.

How the amino acid sequence of a protein determines its three-dimensional structure is a major problem in biology and chemistry. Leading experts in the fields of NMR spectroscopy, X-ray crystallography, protein engineering and molecular modeling offer provocative insights into current views on the protein folding problem and various aspects for future progress.

Protein Folding aims to collect the most important information in the field of protein folding and probes the main principles that govern formation of the three-dimensional structure of a protein from a nascent polypeptide chain, as well as how the functional properties appear. This text is organized into three sections and consists of 15 chapters. After an introductory chapter where the main problems of protein folding are considered at the cellular level in the context of protein biosynthesis, the discussion turns to the conformation of native globular proteins. Definitions and rules of nomenclature are given, including the structural organization of globular proteins deduced from X-ray crystallographic data. Folding mechanisms are tentatively deduced from the observation of invariants in the architecture of folded proteins. The next chapters focus on the energetics of protein conformation and structure, indicating the principles of thermodynamic stability of the native structure, along with theoretical computation studies of protein folding, structure prediction, and folding simulation. The reader is also introduced to various experimental approaches; the reversibility of the unfolding-folding process; equilibrium and kinetic studies; and detection and characterization of intermediates in protein folding. This text concludes with a chapter dealing with problems specific to oligomeric proteins. This book is intended for research scientists, specialists, biochemists, and students of biochemistry and biology.

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 (

Covering experiment and theory, bioinformatics approaches, and state-of-the-art simulation protocols for better sampling of the conformational space, this volume describes a broad range of techniques to study, predict, and analyze the protein folding process. Protein Folding Protocols also provides sample approaches toward the prediction of protein structure starting from the amino acid sequence, in the absence of overall homologous sequences.