Properly folded proteins are necessary for all living organisms. Incorrectly folded proteins can lead to a variety of diseases such as Alzheimer’s Disease or Bovine Spongiform Encephalitis (Mad Cow Disease). Understanding the forces involved in protein folding is essential to the understanding and treatment of protein misfolding diseases. When proteins fold, a significant amount of surface area is buried in the protein interior. It has long been known that burial of hydrophobic surface area was important to the stability of the folded structure. However, the impact of burying polar surface area is not well understood. Theoretical results suggest that burying polar groups decreases the stability, but experimental evidence supports the belief that polar group burial increases the stability. Studies of tyrosine to phenylalanine mutations have shown the removal of the tyrosine OH group generally decreases stability. Through computational investigations into the effect of buried tyrosine on protein stability, favorable van der Waals interactions are shown to correlate with the change in stability caused by replacing the tyrosine with phenylalanine to remove the polar OH group. Two large-scale studies on nearly 1000 high-resolution x-ray structures are presented. The first investigates the electrostatic and van der Waals interactions, analyzing the energetics of burying various atom groups in the protein interior. The second large-scale study analyzes the packing differences in the interior of the protein and shows that hydrogen bonding increases packing, decreasing the volume of a hydrogen bonded backbone by about 1.5 Å3 per hydrogen bond. Finally, a structural comparison between RNase Sa and a variant in which five lysines replaced five acidic groups to reverse the net charge is presented. It is shown that these mutations have a marginal impact on the structure, with only small changes in some loop regions.

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.

Proteins in nature fold to one dominant native structure. Despite being a heavily studied field, predicting the native structure from the amino acid sequence and modeling the folding process can still be considered unsolved problems. In this thesis I present a new approach to this problem with methods borrowed from theoretical physics. In the first part I show how it is possible to use a discrete Frenet frame to define the discrete curvature and torsion of the main chain of the protein. This method is then extended to the side chains as well. In particular I show how to use the discrete Frenet frame to produce a statistical distribution of angles that works in similar fashion as the commonly used Ramachandran plot and side chain rotamers. The discrete Frenet frame displays a gauge symmetry, in the choice of basis vectors on the normal plane, that is reminiscent of features of Abelian-Higgs theory. In the second part of the thesis I show how this similarity with Abelian-Higgs theory can be translated into an effective energy for a protein. The loops of the proteins are shown to correspond to solitons so that the whole protein can be constructed by gluing together any number of solitons. I present results of simulating proteins by minimizing the energy, starting from a real line or straight helix, where the correct native fold is attained. Finally the model is shown to display the same phase structure as real proteins.

Understanding structure and folding of a protein is the key to understanding its biological function and potential role in diseases. Despite the importance of protein folding, a molecular level understanding of this process is still lacking. Solution-state nuclear magnetic resonance (NMR) is a powerful technique to investigate protein structure, dynamics, and folding mechanisms, since it provides residue-specific information. One of the major contributions that govern protein structure appears to be the interaction with the solvent. The importance of these interactions is particularly apparent in membrane proteins, which exist in an amphiphilic environment. Here, individual peptide fragments taken from the disulfide bond forming protein B (DsbB) were investigated in various solvents. The alpha-helical structures that were obtained, suggest that DsbB follows the two-stage model for folding. However, side chains of polar residues showed different conformations compared to the X-ray structure of fulllength protein, implying that polar side-chains may re-orient upon helix packing in order to form the necessary tertiary interactions that stabilize the global fold of DsbB. Model peptides in general represent attractive systems for the investigation of non-covalent interactions important for protein folding, including those with the solvent. NMR structures of the water soluble peptide, BBA5, were obtained in the presence an organic co-solvent, methanol. These structures indicate that the addition of methanol stabilizes an alpha-helix segment, but disrupts a hydrophobic cluster forming a beta-hairpin. Since dynamic effects reduce the ability for experimental observation of individual, bound solvent molecules, results were compared with molecular dynamics simulations. This comparison indicates that the observed effects of NMR structures are due to preferred binding of methanol and reduction of peptide-water hydrogen bonding. NMR structures, such as those determined here, represent a distribution of conformations under equilibrium. The dynamic process of protein unfolding can nevertheless be accessed through denaturation. A method was developed to probe thermal denaturation by measuring the temperature dependence of NOE intensity. Applied to a model peptide, trpzip4, it was confirmed that the beta-hairpin structure of this peptide is stabilized by the hydrophobic cluster formed by tryptophan residues. Together, the peptides investigated here illustrate the important roles that solvent-peptide interactions and side chain-side chain hydrophobic interactions play in forming stable secondary and tertiary structures.

Understanding how proteins fold and bind is interesting since these processes are central to most biological activity. Protein folding and protein-protein interaction are by themselves very complex but using a good and robust system to study them could ease some of the hurdles. In this thesis I have tried to answer some of the fundamental questions of protein folding and binding. I chose to work with PDZ domains, which are protein domains consisting of 90-100 amino acids. They are found in more than 400 human proteins and function mostly as protein-protein interaction units. These proteins are very stable, easy to express and purify and their folding reaction is reversible under most laboratory conditions. I have characterized the interaction of PSD-95 PDZ3 domain with its putative ligand under different experimental conditions and found out that its binding kinetics is sensitive to salt and pH.  I also demonstrated that the two conserved residues R318 and H372 in PDZ3 are responsible for the salt and pH effect, respectively, on the binding reaction. Moreover, I determined that for PSD 95 PDZ3 coupling of distal residues to peptide binding was better described by a distance relationship and there was a very weak evidence of an allosteric network. Further, I showed that another PDZ domain, SAP97 PDZ2 undergoes conformational change upon ligand binding. Also, I characterized the binding mechanism of a dimeirc ligand/PDZ1-2 tandem interaction and showed that despite its apparent complexity the binding reaction is best described by a square scheme. Additionally, I determined that for the SAP 97 PDZ/HPV E6 interaction that all three PDZ domains each bind one molecule of the E6 protein and that a set of residues in the PDZ2 of SAP 97 could operate in an unexpected long-range manner during E6 interaction. Finally, I showed that perhaps all members in the PDZ family could fold via a three state folding mechanism. I characterized the folding mechanism of five different PDZ domains having similar overall fold but different primary structure and the results indicate that all five fold via an intermediate with two transition states. Transition state one is rate limiting at low denaturant concentration and vice versa for transition state two. Comparing and characterizing the structures of the transition states of two PDZ domains using phi value analysis indicated that their early transition states are less similar as compared to their late transition states.

Proteins are important building blocks of all living organisms. They are composed of a defined sequence of different amino acids, and fold into a specific three-dimensional, ordered structure. The three-dimensional structure largely determines the function of the protein, but protein function always requires motion. Small movements within the protein structure govern the functional properties, and this thesis aims to better understand these discrete protein movements. The motions within the protein structure are governed by thermodynamics, which therefore is useful to predict protein interactions. Nuclear magnetic resonance (NMR) is a powerful tool to study proteins at atomic resolution. Therefore, NMR is the primary method used within this thesis, along with other biophysical techniques such as Fluorescence spectroscopy, Circular Dichroism spectroscopy and in silico modeling. In paper I, NMR in combination with molecular engineering is used to show that the folding of the catalytical subdomains of the enzyme Adenylate kinase does not affect the core of the protein, and thus takes a first step to linking folding, thermodynamic stability and catalysis. In paper II, the structure of the primary allergen from Brazil nut, Ber e 1, is presented along with biophysical measurements that help explain the allergenic potential of the protein. Paper III describes the need for a specific Brazil nut lipid fraction needed to induce an allergenic response. NMR and fluorescence spectroscopy is used to show that there is a direct interaction between Ber e 1 and one or several components in the lipid fraction.

Proteins present at the synapse need to be multitasking in order to perform all vital functions in this limited space. In this thesis I have analyzed the function and evolution of such proteins, focusing on the PDZ domain and the paralemmin family. The PDZ domains bind to a wide variety of interaction partners. The affinity for each partner is regulated by residues at the binding site, but also through intradomain allostery. How this intradomain allostery is transferred to the binding site is not established. I here show that side chain interactions can explain all transfer of intradomain allostery in three analyzed PDZ domains. A circularly permuted PDZ domain has an identical set of amino acids as the original protein and a very similar structure with only a few perturbed side chains. By using the circular permutant I show that a slight alteration in the position of a side chain leads to a corresponding change in allosteric signal. I further study the folding of several PDZ domains and show that they all fold via a conserved folding mechanism, supporting the notion that the final structure has a part in deciding folding mechanism. The folding mechanism of the circularly permuted PDZ domain is conserved compared to the original protein illustrating how circular permutations can be tolerated through evolution. The multifunctionality of paralemmins probably lies in their highly flexible structures. I have studied the evolution of the paralemmins and found that the four mammalian paralemmins arose in the two whole-genome duplications that occurred early in the vertebrate evolution. The fact that all four paralemmins have survived evolution since the gene duplications suggests that they have important functions, possibly in the development of the nervous system. Synaptic proteins are crucial for many biological processes, and their misfolding implicated in many diseases. The results presented here shed light on the mechanisms of action of the synaptic proteins and will help us to understand how they generate disease.

Misfolding of membrane proteins as a result of mutations that disrupt their functions in substrate transport across the membrane or signal transduction is the cause of many significant human diseases. Yet, we still have a limited understanding of the direct consequences of these mutations on folding and function - a necessary step toward the rational design of corrective therapeutics. This thesis addresses the gap in understanding the residue-specific implications for folding through a series of experiments that utilize the cystic fibrosis transmembrane conductance regulator (CFTR) as a model in various contexts. We first examined the thermodynamic implications of mutations in the soluble nucleotide binding domain 1 (NBD1) of CFTR. We found that mutations can have a significant effect on thermodynamic stability that is masked in non-physiological conditions. Our studies were then focussed on a membrane-embedded hairpin CFTR fragment comprised of transmembrane segments 3 (TM3) and 4 (TM4) to evaluate the direct effects of mutations on folding in a systematic manner. It was found that the translocon-mediated membrane insertion of helices closely parallels a basic hydrophobic-aqueous partitioning event. This study was then extended to determine residue-specific effects on helix-helix association. We found that this process is not solely dependent on hydropathy, but there is a context dependence of these results with regard to residue position within the helix. Overall, these findings constitute a key step in relating mutation-derived effects on membrane protein folding to the underlying basis of human disease such as cystic fibrosis.

Recombinant human macrophage colony stimulating factor beta (rhm-CSFβ) is a glycoprotein that stimulates the proliferation, differentiation and survival of cells belonging to the monocyte-macrophage lineage. It contains nine inter-subunit and intra-subunit disulfide bonds and represents an excellent model system for studying disulfide bond formation during protein folding because the assembly of its monomeric subunits and the maturation of its biological activity depend on the progressive formation of the correct disulfide structure during in vitro folding. Knowledge obtained from these studies can be potentially useful in understanding the roles of disulfide bond formation during protein folding in general. rhm-CSF8 was modified by partial reduction of disulfide bonds, yielding CN¹⁵⁷'¹⁵⁹-modified rhm-CSFβ. The modification did not affect the biological activity, stability, or the overall conformation of the protein. However, the C-terminal regions near the modification sites were shown to exhibit faster deuterium exchange behavior as a result of the chemical modification, indicating that the C-terminal regions became more flexible. Folding kinetics of rhm-CSFβ and CN¹⁵⁷'¹⁵⁹-modified rhm-CSFβ were shown to be essentially the same, suggesting that the modification did not affect the folding kinetics of the oxidized rhm-CSFβ. The denatured and reduced rhm-CSFβ was refolded with the aid of a chemical oxidant. The data indicated that the in vitro folding rhm-CSFβ proceeded via multiple pathways involving monomeric and dimeric intermediates. Disulfide bond shuffling catalyzed by GSH/GSSG represented an important isomerization step in folding. A dimeric intermediate, D-SS8-cam2, was isolated and identified as a kinetic trap, perhaps requiring significant structural arrangement to convert to the native protein. The heterogeneous folding mixture detected by both disulfide bond quenching and H/D pulsed labeling indicate that rhm -CSFβ folding is a diffusion like process as described by the folding funnel model. / Graduation date: 2003

We show that a single-copy tempering method is useful in protein-folding simulations of large scale and high accuracy (explicit solvent, atomic representation, and physics-based potential). The method uses a runtime estimate of the average potential energy from an integral identity to guide a random walk in the continuous temperature space. It was used for folding three mini-proteins, trpzip2 (PDB ID: 1LE1), trp-cage (1L2Y), and villin headpiece (1VII) within atomic accuracy. Further, using a modification of the method with a dihedral bias potential added on the roof temperature, we were able to fold four larger helical proteins: α3D (2A3D), α3W (1LQ7), Fap1-NRα (2KUB) and S-836 (2JUA). We also discuss how to optimally use simulation data through an integral identity. With the help of a general mean force formula, the identity makes better use of data collected in a molecular dynamics simulation and is more accurate and precise than the common histogram approach.