Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 95-126.

Native states of globular proteins typically show stabilization in the range of 5 to 15 kcal/mol with respect to their unfolded states. There has been a considerable progress in the area of protein stability and folding in recent years, but increasing protein stability through rationally designed mutations has remained a challenging task. Current ability to predict protein structure from the amino acid sequence is also limited due to the lack of quantitative understanding of various factors that defines the single lowest energy fold or native state. The most important factors, which are considered primarily responsible for the structure and stability of the biological active form of proteins, are hydrophobic interactions, hydrogen bonding and electrostatic interactions such as salt bridges as well as packing interactions. Several studies have been carried out to decipher the importance of each these factors in protein stability and structure via rationally designed mutant proteins. The limited success of previous studies emphasizes the need for comprehensive studies on various aspect of protein stability. An integrated approach involving thermodynamic and structural analysis of a protein is very useful in understanding this particular phenomenon. This approach is very useful in relating the thermodynamic stability with the structure of a protein. A survey of the current literature on thermodynamic stability of protein indicates that the majority of the model proteins which have been used for understanding the determinants of protein stability are small, monomeric, single domain globular proteins like RNase A, Lysozyme and Myoglobin. On the other hand large proteins often show complex unfolding transition profiles that are rarely reversible. The major part of this thesis is focused on studying potential stabilizing/destabilizing interactions in small and large globular proteins. These interactions have been identified and characterized by exploring the effects of various rationally designed mutations on protein stability. Spectroscopic, molecular biological and calorimetric techniques were employed to understand the relationships between protein sequence, structure and stability. The experimental systems used are Leucine binding proteins, Leucine isoleucine valine binding protein (LIVBP), Maltose binding protein (MBP), Ribose binding protein (RBP) and Thioredoxin (Trx). The last section of the thesis discusses thermodynamic properties of molten globule states of the periplasmic protein LBP, LIVBP, MBP and RBP. The amino acid Pro is unique among all the twenty naturally occurring amino acids. In the case of proline, the Cδ of the side chain is covalently linked with the main chain nitrogen atom in a five membered ring. Therefore, Pro lacks amide hydrogen and it is not able to form a main chain hydrogen bond with a carbonyl oxygen. Hence Pro is typically not found in the hydrogen bonded, interior region of α-helix. There have been several studies which showed that introduction of the Pro residue into the interior of an α-helix is destabilizing. Although, it is not common to find Pro residue in the interiors of an α-helix, it has been reported that it occurs with appreciable frequency (14%). The thermodynamic effects of replacements of Pro residue in helix interiors of MBP were investigated in Chapter 2 of this thesis. Unlike many other small proteins, MBP contains 21 Pro residues distributed throughout the structure. It contains three residues in the interiors of α-helices, at positions 48, 133 and 159. These Pro residues were replaced with an alanine and serine amino acids using site directed mutagenesis. Stabilities of all the mutant and wild type proteins have been studied via isothermal chemical denaturation at pH 7.4 and thermal denaturation as a function of pH ranging from pH 6.5 to 10.4. It has been observed that replacement of a proline residue in the middle of an α-helix does not always stabilize a protein. It can be stabilizing if the carbonyl oxygen of residue (i-3) or (i-4) is well positioned to form a hydrogen bond with the ith (mutated) residue and the position of mutation is not buried or conserved in the protein. Partially exposed position have the ability to form main chain hydrogen bonds and Ala seems to be a better choice to substitute Pro than Ser. Unlike other amino acids, the pyrolidine ring of Pro residue imposes rigid constraints on the rotation about the N---Cα bond in the peptide backbone. This causes conformational restriction of the φ dihedral angle of Pro to -63±15º in polypeptides. Therefore, introduction of a rigid Pro residue into an appropriate position in a protein sequence is expected to decrease the conformational entropy of the denatured state and consequently lead to protein stabilization. In Chapter 3 of this thesis, the thermodynamic effects of Pro introduction on protein stability has been investigated in LIVBP, MBP, RBP and Trx. Thirteen single and two double mutants have been generated in the above four proteins. Three of the MBP mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, CD spectroscopy was used to show the absence of structural changes. Stability of all the mutant and wild type proteins was studied via isothermal chemical denaturation at neutral pH and thermal denaturation as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, six of the thirteen single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, two mutants showed no change and five were destabilized. In five of the six cases, the stabilization was because of a reduced entropy of unfolding. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were non-additive. In addition to the hydrogen bond, hydrophobic and electrostatic interactions, other interactions like cation-π and aromatic-aromatic interactions etc. are also considered to make important contributions to protein stability. The relevance of cation-π interaction in biological systems has been recognized in recent years. It has been reported that positively charged amino acids (Lys, Arg and His) are often located within 6 Å of the ring centroids of aromatic amino acids (Phe, Tyr and Trp). The importance of cation-π interaction in protein stability has been suggested by previous theoretical and experimental studies. We have attempted to determine the magnitude of cation-π interactions of Lys with aromatic amino acids in four different proteins (LIVBP, MBP, RBP and Trx) in Chapter 4 of the thesis. Cation-π pairs have been identified by using the program CaPTURE. We have found thirteen cation-π pairs in five different proteins (PDB ID’s 2liv, 1omp, 1anf, 1urp and 2trx). Five cation-π pairs were selected for the study. In each pair, Lys was replaced with Gln and Met. In a separate series of experiments, the aromatic amino acid in each cation-π pair was replaced by Leu. Stabilities of wild type (WT) and mutant proteins were characterized by similar methods, to those discussed in previous chapters. Gln and Aromatic → Leu mutants were consistently less stable than the corresponding Met mutants reflecting the non-isosteric nature of these substitutions. The strength of the cation-π interaction was assessed by the value of the change in the free energy of unfolding (ΔΔG0=ΔG0 (Met) - ΔG0(WT)). This ranged from +1.1 to –1.9 kcal/mol (average value – 0.4 kcal/mol) at 298 K and +0.7 to –2.6 kcal/mol (average value –1.1 kcal/mol) at the Tm of each WT. It therefore appears that the strength of cation-π interactions increases with temperature. In addition, the experimentally measured values are appreciably smaller in magnitude than the calculated values with an average difference |ΔG0expt -ΔG0calc|avg of 2.9 kcal/mol. At room temperature, the data indicate that cation-π interactions are at best weakly stabilizing and in some cases are clearly destabilizing. However at elevated temperatures, close to typical Tm’s, cation-π interactions are generally stabilizing. In Chapter 5, we have attempted to characterize molten globule states for the periplasmic proteins LBP, LIVBP, MBP and RBP. It was observed that all these proteins form molten globule states at acidic pH (3 - 3.4). All these molten globule states showed cooperative thermal transitions and bound with their ligand comparable to (LBP and LIVBP) or with lower (MBP and RBP) affinity than the corresponding native states. Trp, ANS fluorescence and near-UV CD spectra for ligand bound and free forms of molten globule states were found to be very similar. This shows that molten globule states of these proteins have the ability to bind to their corresponding ligand without conversion to the native state. All four molten globule states showed destabilization relative to the native state. ΔCp values indicate that these molten globule states contain approximately 29-67% of tertiary structure relative to the native state. All four proteins lack prosthetic groups and disulfide bonds. Therefore, it is likely that molten globule states of these proteins are stabilized via hydrophobic and hydrogen bonding interactions.

<p>Gene expression is regulated in response to metabolic necessities and environmental changes throughout the life of a cell.</p><p>A major part of this regulation is governed at the level of transcription, deciding whether messengers to specific genes are produced or not.</p><p>This decision is triggered by the action of transcription factors, proteins which interact with specific sites on DNA and thus influence the rate of transcription of proximal genes.</p><p>Mapping the organisation of these transcription factor binding sites sheds light on potential causal relations between genes and is the key to establishing networks of genetic interactions, which determine how the cell adapts to external changes.</p><p>In this work I review briefly the basics of genetics and summarise popular approaches to describe transcription factor binding sites, from the most straight forward to finally discuss a biophysically motivated representation based on the estimation of free energies of molecular interactions.</p><p>Two articles on transcription factors are contained in this thesis, one published (Aurell, Fouquier d'Hérouël, Malmnäs and Vergassola, 2007) and one submitted (Fouquier d'Hérouël, 2008).</p><p>Both rely strongly on the representation of binding sites by matrices accounting for the affinity of the proteins to specific nucleotides at the different positions of the binding sites.</p><p>The importance of non-specific binding of transcription factors to DNA is briefly addressed in the text and extensively discussed in the first appended article:</p><p>In a study on the affinity of yeast transcription factors for their binding sites, we conclude that measured in vivo protein concentrations are marginally sufficient to guarantee the occupation of functional sites, as opposed to unspecific emplacements on the genomic sequence.</p><p>A common task being the inference of binding site motifs, the most common statistical method is reviewed in detail, upon which I constructed an alternative biophysically motivated approach, exemplified in the second appended article.</p>

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Call number: LD2668 .T4 1955 L67 / Master of Science

In the present study non-5-HT₁(A)/non-5-HT₁(C) binding sites in the rabbit caudate nucleus (CN) were examined to determine if they might be identical to the recently discovered 5-HT₁(D) sites in the bovine CN. The characterizations were carried out measuring high-affinity [³H]5-HT binding under conditions where 5-HT₁(A) and 5-HT₁(C) sites were pharmacologically masked in both tissues. Comparison of the pharmacologic profiles of the bovine 5-HT₁(D) and rabbit non-5-HT₁(A)/non-5-HT₁(C) sites revealed similarities, but showed distinct differences. [³H]5-HT binding in the bovine CN was significantly more sensitive to inhibition by GTP than was [³H]5-HT binding in the rabbit CN, and this effect was differentially sensitive to calcium and other divalent cations (i.e., Mg²⁺, Mn²+)⁺in the two tissues. [³H]5-HT binding in the bovine CN was significantly more sensitive to inhibition by NEM than it was in the rabbit CN. Thus, it may be concluded that the non-5-HT₁(A)/non-5-HT₁(C) [³H]5-HT binding sites in rabbit CN are distinct from those in the bovine CN, and we propose that they be tentatively identified as 5-HT₁(R) to distinguish them from the 5-HT₁(D) site.

AreA, a 96 kDa gene regulatory protein involved in nitrogen metabolite repression in Aspergillus nidulans, is a member of the GATA family of zinc finger DNA binding proteins, and regulates the expression of around 100 genes. This project was designed to examine the domain structure of AreA in this region of the protein, and to characterise the DNA binding domain Limited proteolysis has been employed to identify structural domains in the Cterminal region of AreA, which has been cloned and over-produced in E.coli. A variety of proteases have been used, and each reveals a dominant stable fragment of approximately 17-22 kDa. N-terminal sequencing and mass spectroscopy have been used to identify a number of these fragments. The major product following limited proteolysis by Glu-C is composed of two closely related species, a 164 residue fragment (17,489 Da) and a 157 residue fragment (16,857 Da). Both fragments encompass the Zn-finger motif, and share the same Cterminus, differing at the N-terminus by only 7 amino acids. The DNA sequence coding for the 157 residue fragment (16,857 Da) has been cloned and over-produced as a His-tag fusion protein. Further studies on this domain have shown that this putative domain has a relatively strong DNA binding constant with values in the nanomolar range. Structural analysis using Circular Dichroism, NMR and fluorescence suggests that the domain contains some irregular or unstructured regions. The regions that are structured are likely to be from the zinc-finger region, since DNA binding is maintained.

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