To understand protein stability and the mechanism of protein folding, it is essential that we gain a better understanding of the ensemble of conformations that make up the denatured state of a protein. The primary goal of the research described here was to see what we might learn about the denatured state using fluorescence. To this end, tryptophan was introduced at five sites in Ribonuclease Sa (RNase Sa): D1W, Y52W, Y55W, T76W, and Y81W. The fluorescent properties of the denatured states of these five proteins were studied and compared to the fluorescent properties of eight model compounds: N-acetyl-tryptophan-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and five pentapeptides based on the sequence around the original tryptophan substitutions in RNase Sa. Regardless of the denaturant, λmax for the proteins and model compounds differed very little, 349.3 ± 1.2 nm. However, significant differences were observed in the fluorescence intensity at λmax (IF), suggesting that IF is more sensitive to the immediate environment than λmax. The differences in IF are due in part to quenching by neighboring side chains. More importantly, IF was always significantly greater in the protein than in its corresponding pentapeptide, indicating that the protein exerts an effect on the tryptophan, which cannot be mimicked by the pentapeptide models. Acrylamide and iodide quenching experiments were also performed on the model compounds and proteins. Significant differences in the Stern-Volmer quenching constant (KSV) were also observed between the proteins and between the proteins and their corresponding pentapeptides. Importantly, the KSV for the protein was always less than in its corresponding pentapeptide. These data along with the IF data show that non-local structure in the unfolded state influences tryptophan fluorescence and accessibility. In summary, these and our other studies show that fluorescence can be used to gain a better understanding of the denatured states of proteins.

In this thesis, the folding process of the de novo designed polypeptide chignolin was elucidated through atomic-scale Molecular Dynamics (MD) computer simulations. In a series of long timescale and replica exchange MD simulations, chignolin’s folding and unfolding was observed numerous times and the native state was identified from the computed Gibbs free-energy landscape. The rate of the self-assembly process was predicted from the replica exchange data through a novel algorithm and the structural fluctuations of an enzyme, lysozyme, were analyzed. DNA’s structural flexibility was investigated through experimental structure determination methods in the liquid and gas phase. DNA nanostructures could be maintained in a flat geometry when attached to an electrostatically charged, atomically flat surface and imaged in solution with an Atomic Force Microscope. Free in solution under otherwise identical conditions, the origami exhibited substantial compaction, as revealed by small angle X-ray scattering. This condensation was even more extensive in the gas phase. Protein folding is highly reproducible. It can rapidly lead to a stable state, which undergoes moderate fluctuations, at least for small structures. DNA maintains extensive structural flexibility, even when folded into large DNA origami. One may reflect upon the functional roles of proteins and DNA as a consequence of their atomic-level structural flexibility. DNA, biology’s information carrier, is very flexible and malleable, adopting to ever new conformations. Proteins, nature’s machines, faithfully adopt highly reproducible shapes to perform life’s functions robotically.

Protein folding is the process during which an extended and unstructured polypeptide converts to its compact folded structure that is most often the functional state. The process has been characterized extensively in dilute buffer in-vitro during the last decades but the actual biological place for this process is the inside of living cells. The cytoplasm of a cell is filled with a plethora of different macromolecules that together occupy up to 40% of the total volume. This large amount of macromolecules restricts the available space to each individual molecule, which has been termed macromolecular crowding. Macromolecular crowding results in excluded volume effects and also increases chances for non-specific interactions. Macromolecular crowding should favor reactions that lead to a decrease in the total occupied volume by all molecules, such as folding reactions. Theoretical models have predicted that the stability of protein folded states should increase in presence of macromolecular crowding due to unfavorable effects on the extended unfolded state. To understand protein folding and function in living systems, we need to have a defined quantitative link between in-vitro dilute conditions (where most biophysical experiments are made) and in-vivo crowded conditions. An important question is thus how macromolecular crowding modifies the biophysical properties of a protein. The work underlying this thesis focused on how macromolecular crowding tunes protein equilibrium stability and kinetic folding processes. To mimic the crowded cellular environment, synthetic sugar-based polymers (Dextrans of different sizes and Ficoll 70) were used as crowding agents (crowders) in controlled in-vitro experiments. In contrast to previous studies which often have focused on one protein and one crowder at a time, the goal here was to make systematic analyses of how size, shape and concentration of the crowders affect both equilibrium and kinetic properties of structurally-different proteins. Three model proteins (cytochrome c, apoazurin and apoflavodoxin) were investigated under crowding by Ficoll 70 and different-size Dextrans, using various spectroscopic techniques such as far-UV circular dichroism and intrinsic tryptophan fluorescence. Thermodynamic models were applied to explain the experimental results. It was discovered that equilibrium stability of all three proteins increased in presence of crowding agents in a crowder concentration dependent manner. The stabilization effect was around 2-3 kJ/mol, larger for the various Dextrans than for Ficoll 70 at the same g/l, but independent of Dextran size (in the range 20 to 70 kDa). To further investigate the cause for the stabilization a theoretical crowding model was applied. In this model, Dextran and Ficoll were modeled as elongated rods and the protein was represented as a sphere, where the folded sphere representation was smaller than the unfolded sphere representation. It is notable that the observed stability changes could be reproduced by this model taking only steric interactions into account. This correlation showed that when using sugar-based crowding agents, excluded volume effects could be studied in isolation and there were no contributions from nonspecific interactions. Time-resolved experiments with apoazurin and apoflavodoxin revealed an increase in the folding rate constants while the unfolding rates were invariant in the presence of crowding agents. For apoflavodoxin and cytochrome c, the presence of crowding agents also altered the folding pathway such that it became more homogeneous (cytochrome c) and it gave less misfolding (apoflavodoxin). These results showed that macromolecular crowding restricts the conformational space of the unfolded polypeptide chain, makes the conformations more compact which, in turn, eliminates access to certain pathways. The results from kinetic and equilibrium measurements on three model proteins, together with available data from the literature, demonstrate that macromolecular crowding effects due to volume exclusion are in the order of a few kJ/mol. Considering the numerous concentration balances and cross-dependent reactions of the cellular machinery, small changes in energetics/kinetics of the magnitudes found here can still have dramatic consequences for cellular fitness. In fact local and transient changes in macromolecular crowding levels may be a way to tune biochemical reactions without invoking gene expression.

This thesis is an investigation of the role of the prosegment (PS) of pepsinogen in the binding, refolding and inhibition of pepsin. Native pepsin (Np) is irreversibly denatured, and folds to a stable, non-native state under refolding conditions, termed refolded pepsin (Rp) (Dee and Yada 2010). When added separately, the PS binds Rp, catalyzes folding to the native-like state and inhibits Np (Dee and Yada 2010). It was hypothesized, owing to the high sequence conservation, that N-terminal PS residues are critical to PS catalyzed folding. Synthetic peptides of N-terminal truncations (N16, N29), C-terminal truncations (C15, C28), and full length, wild-type (Wt) PS were examined. N-terminal residues were required for binding to Rp and catalyzing folding, while both N29 and C28 truncations had similar inhibition constants. Remarkably, the foldase activity of N-terminal truncation (N29) was only 2.5 fold slower than Wt, supporting that PS foldase activity is stored almost entirely within the highly conserved N29 region.

No description available.

Threefoil is a small engineered protein of 141 amino acids which is a member of the beta-trefoil superfamily, with three-fold symmetry and high thermal and kinetic stability. Its primary sequence was designed based on a predicted beta-trefoil glycosidase from the halophilic Archaeon Haloarcula marismortui. Threefoil predominantly forms inclusion bodies when over-expressed in Escherichia coli at 37??C, with little to no protein soluble in the cytoplasm. Nevertheless, Threefoil is capable of adopting a native beta-trefoil structure when refolded from solubilized inclusion bodies. The focus of this thesis is on characterization of the folding and stability of Threefoil through thermodynamic and kinetic experiments for wild-type Threefoil, in addition to sugar- and metal-binding studies and characterization of Threefoil inclusion bodies. Various Threefoil mutants, designed to increase protein stability, are also characterized to probe the origins of, as well as to give insight into, the mechanism of inclusion body formation. The thermodynamic and kinetic stability of wild-type Threefoil was studied using spectral probes, mainly fluorescence, circular dichroism (CD) and dynamic light scattering (DLS). The major observed spectral changes in kinetic and thermodynamic experiments can be fit to a 2-state transition between the folded state and a denatured state containing extensive residual secondary structure. At high protein concentrations, the folding of wild-type Threefoil is complicated by protein misfolding and aggregation. As Threefoil is remarkably resistant to denaturation even at high concentrations of urea and guanidine hydrochloride (GuHCl), studies were also conducted in guanidine isothiocyanate (GuSCN), which is a much stronger denaturant than urea and GuHCl. Remarkably, the time that is required for Threefoil samples to reach equilibrium in renaturation curves is approximately 100 days, while equilibrium by denaturation in the stronger denaturant, GuSCN, requires more than two years. The expression levels of Threefoil mutants A62V, Q78I, D85P and D93P were also studied. None of the four mutants studied exhibited any pronounced increase in solubility compared to wild-type when expressed in E. coli.

We study the collapse transition of a lattice based protein model including an explicit coarse-grained model of a solvent. This model accounts for explicit hydrophobic interactions, and it is studied by Monte Carlo simulation. The protein is modelled as self-avoiding random walk with nearest neighbor interactions on a two dimensional lattice. Without the solvent, universal quantities of the chain around the collapse transition temperature are well known. Hydrophobicity is then modelled through a lattice of solvent molecules in which each molecule can have Q states depending of an orientation variable. Only one state is energetically favored, when two neighboring solvent molecules are both in the same state of orientation. The monomers are placed in interstitial position of the solvent lattice, and are only allowed to occupy sites surrounded by solvent cells of the same orientation. The potential of mean force between two interstitial solute molecules is calculated, showing a solvent mediated attraction typical of hydrophobic interactions. We then show that this potential increases with the energy of hydrogen bond formation as it appears in the model, while its characteristic range decreases. More importantly, we show that the chain embedded in the solvent undergoes a collapse transition, with the temperature of the transition being shifted relative to that of the chain in isolation. We calculate several critical exponents near the collapse transition, and we observe that their values are not conserved in presence of the explicit solvent.

Single-molecule fluorescence spectroscopy techniques such as Fluorescence Correlation Spectroscopy (FCS) and single-molecule Förster Resonance Energy Transfer (smFRET) not only possess an unprecedented high sensitivity but also have high temporal and spatial resolution. Therefore, they have an immense potential both in investigation of fundamental biological principles and in clinical applications. FCS analyses are based on both theoretical approximations of the beam geometry and assumptions of the underlying molecular processes. To address the accuracy of analysis, firstly the experimental conditions that should be fulfilled in order to obtain reliable physical parameters are discussed and the input parameters are carefully controlled accordingly to demonstrate the performance of FCS measurements on our home-built confocal multiparameter photon-counting microscope in several in vitro and in-vivo applications. Secondly, we performed a comprehensive FCS analysis of rhodamine family of dyes to evaluate the validity of assigning the correlation relaxation times to the time constant of conformational dynamics of biomolecules. While it is the common approach in literature our data suggests that conformational dynamics mainly appear in the correlation curve via modulation of the dark states of the fluorophores. The size and shape of the folded, unfolded and chemically-denatured states of the N-terminal Src-homology-3 of downstream of receptor kinases (DrkN SH3) were investigated by FCS and smFRET burst experiments. Based on the data, we conclude that a considerable sub-population of the denatured protein is in a closed loop state which is most likely formed by cooperative hydrogen bonds, salt bridges and nonpolar contacts. As a clinical application, we developed and characterized an ultrasensitive capillary electrophoresis method on our multiparameter confocal microscope. This allowed us to perform Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) with about 500 times better sensivity than a commercial instrument. Quite remarkably, we were able to analyze samples of cell lysate down to the contents of a single cell.

SH3 domains are excellent models for probing folding and protein interactions. This thesis describes NMR studies of several SH3 domains, including the N-terminal SH3 domain of the Drosophila adaptor protein Drk (drkN SH3 domain), the SH3 domain of the proto-oncogene tyrosine-kinase Fyn, and the SH3 domains of the human adaptor protein CIN85, involved in Cbl-mediated downregulation of epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs). The drkN SH3 domain is an ideal system for studying disordered states. The unique quality of this isolated domain is that it exists in an approximately 50/50 equilibrium between its folded and unfolded states under non-denaturating buffer conditions. Interestingly, the single T22G mutation dramatically stabilizes the domain. Here the NMR structures of the drkN SH3 domain and its T22G mutant are determined and compared in order to illuminate the causes of the marginal stability of the domain. Solvent exposure of the folded and the unfolded drkN SH3 domains are probed and compared with a novel NMR technique using molecular oxygen dissolved in solution as a paramagnetic probe. The changes in partial molar volume along the folding trajectories of the drkN SH3 and Fyn SH3 domains are also studied and analyzed here in terms of changes in protein hydration and packing accompanying folding. Finally, the interactions between the SH3 domains of CIN85 and ubiquitin are discussed. All three are shown to bind ubiquitin. The structure of the SH3-C domain in complex with ubiquitin is presented and the effect of disruption of ubiquitin binding on ubiquitination of CIN85 and EGFR in vivo is discussed. SH3 domains are easily amendable to a wide range of NMR approaches and provide a good system for development and testing of novel methods. Through the use of these approaches significant insights into details of SH3 domain structure, stability, mechanisms of folding and cellular function have been gained.

The AAA+ chaperone Hsp104 mediates the reactivation of aggregated proteins in Saccharomyces cerevisiae and is crucial for cell survival after exposure to stress. Protein disaggregation depends on cooperation between Hsp104 and a cognate Hsp70 chaperone system. Hsp104 forms a hexameric ring with a narrow axial channel penetrating the centre of the complex. In Chapter 2, I show that conserved loops in each AAA+ module that line this channel are required for disaggregation and that the position of these loops is likely determined by the nucleotide bound state of Hsp104. This evidence supports a common protein remodeling mechanism among Hsp100 members in which proteins are unfolded and threaded along the axial channel. In Chapter 3, I use a peptide-based substrate mimetic to reveal other novel features of Hsp104’s disaggregation mechanism. An Hsp104-binding peptide selected from solid phase arrays recapitulated several properties of an authentic Hsp104 substrate. Inactivation of the pore loops in either AAA+ module prevented stable peptide or protein binding. However, when the loop in the first AAA+ was inactivated, stimulation of ATPase turnover in the second AAA+ module of this mutant was abolished. Drawing on these data, I propose a detailed mechanistic model of protein unfolding by Hsp104 in which an initial unstable interaction involving the loop in the first AAA+ module simultaneously promotes penetration of the substrate into the second axial channel binding site and activates ATP turnover in the second AAA+ module. In Chapter 4, I explore the recognition elements within a model Hsp104-binding peptide that are required for rapid binding to Hsp104. Removal of bulky hydrophobic residues and lysines abrogated the ability of this peptide to function as a peptide-based substrate mimetic for Hsp104. Furthermore, rapid binding of a model unfolded protein to Hsp104 required an intact N-terminal domain and ATP binding at the first AAA+ module. Taken together, I have defined numerous structural features within Hsp104 and its model substrates that are crucial for substrate binding and processing by Hsp104. This work provides a theoretical framework that will encourage research in other protein remodeling AAA+ ATPases.