The Mediator complex was originally discovered in the yeast Saccharomyces cerevisiae and has since then been shown to be required for transcriptional regulation both in vitro and in vivo. The Mediator complex also stimulates basal, unregulated transcription and serves as a bridge by conveying signals from promoter-bound transcriptional regulatory proteins such as activators and repressors to the RNA Polymerase II general transcriptional machinery. The Mediator consists of 21 subunits and can be divided into three distinct modules head, middle and tail. Despite the tremendous progress that has been achieved so far in characterizing the Mediator complex both functionally and structurally, many aspects of the complex are not yet well understood. The objective of this work is to achieve further understanding of the Mediator complex by studying the folding of different protein subunits, their interactions and how that affects assembly of the Mediator complex. In our first study we made a temperature-sensitive med21 mutant and used it to identify genes that can suppress the mutation when present in high copy number. Among the 10 genes that we identified, the strongest suppressors were Med7 and Med10, which encode Mediator subunits, and Ash1, which encodes a repressor of the HO gene. We also used 2-hybrid experiments and immunoprecipitation to study protein-protein interactions between Med21 and the Med4, Med7 and Med10 proteins which are all essential for viability and located within the middle domain of the Mediator complex. We found that the N-terminal 2-8 amino acids of Med21 are required for interactions with Med7 and Med10. These results led us to propose a model in which the N-terminal part of Med21 functions as a molecular switchboard where competing signals from various activators, repressors and mediator subunits are integrated prior to reaching the general transcription machinery. In our second study, we extended our studies of protein-protein interactions to another part of the mediator complex by studying the folding and the assembly processes of the mediator head domain subunits Med8, Med18 and Med20. Using purified proteins and a combination of several different methods such as immunoprecipitation, far-UV circular dichroism and fluorescence, we demonstrated that the Med8, Med18 and Med20 subunits are interdependent on each other for proper folding and complex formation.

Proper folding of a protein to its native state is critical for the protein to be fully functional under biological conditions. Understanding protein folding and protein folding evolution within the same structural family are key to understand which processes assist or hinder protein folding and how to prevent misfolding. Tm0979 from Thermotoga maritima, Mth1491 from Methanobacterium thermoautotrophicum and YchN from Escherichia coli belong to the homologous superfamily of YchN-like proteins (SCOP and CATH: 3.40.1260.10). The structures of these proteins have been solved as part of structural proteomics projects, which consist of solving protein structures on a genome wide scale. In solution, Tm0979 forms a homodimer whereas Mth1491 folds as a trimer and YchN is a homohexamer. The structures of the individual monomeric subunits of these three proteins have high structural similarity, despite very low sequence similarity. The biological roles of these proteins are not yet well defined, but seem to be involved in catalysis of sulphur redox reactions. This thesis focuses on characterisation of the Tm0979 homodimer and the Mth1491 homotrimer, as well as the determination of the folding mechanisms of these two proteins. The folding mechanisms of the proteins are compared to each other and to the mechanisms of other dimeric and trimeric proteins. The evolution and basis of oligomeric structure within the YchN family are analyzed. Mutations of Tm0979 and Mth1491 are designed as a basis for future work to investigate processes responsible for switches in oligomeric protein quaternary.structure.

Proper folding of a protein to its native state is critical for the protein to be fully functional under biological conditions. Understanding protein folding and protein folding evolution within the same structural family are key to understand which processes assist or hinder protein folding and how to prevent misfolding. Tm0979 from Thermotoga maritima, Mth1491 from Methanobacterium thermoautotrophicum and YchN from Escherichia coli belong to the homologous superfamily of YchN-like proteins (SCOP and CATH: 3.40.1260.10). The structures of these proteins have been solved as part of structural proteomics projects, which consist of solving protein structures on a genome wide scale. In solution, Tm0979 forms a homodimer whereas Mth1491 folds as a trimer and YchN is a homohexamer. The structures of the individual monomeric subunits of these three proteins have high structural similarity, despite very low sequence similarity. The biological roles of these proteins are not yet well defined, but seem to be involved in catalysis of sulphur redox reactions. This thesis focuses on characterisation of the Tm0979 homodimer and the Mth1491 homotrimer, as well as the determination of the folding mechanisms of these two proteins. The folding mechanisms of the proteins are compared to each other and to the mechanisms of other dimeric and trimeric proteins. The evolution and basis of oligomeric structure within the YchN family are analyzed. Mutations of Tm0979 and Mth1491 are designed as a basis for future work to investigate processes responsible for switches in oligomeric protein quaternary.structure.

Proteins need fold to perform their biological function. Thus, understanding how proteins fold could be the key to understanding life. In the first study, the stability and structure of several !-hairpin peptide variants derived from the C-terminus of the B1 domain of protein G (PGB1) were investigated by a number of experimental and computational techniques. Our analysis shows that the structure and stability of this hairpin can be greatly affected by one or a few simple mutations. For example, removing an unfavorable charge near the N-terminus of the peptide (Glu42 to Gln or Thr) or optimization of the N-terminal charge-charge interactions (Gly41 to Lys) both stabilize the peptide, even in water. Furthermore, a simple replacement of a charged residue in the turn (Asp47 to Ala) changes the !-turn conformation. Our results indicate that the structure and stability of this !?hairpin peptide can be modulated in numerous ways and thus contributes towards a more complete understanding of this important model !-hairpin as well as to the folding and stability of larger peptides and proteins. The second study revealed that PGB1 and its variants can form amyloid fibrils in vitro under certain conditions and these fibrils resemble those from other proteins that have been implicated in diseases. To gain a further understanding of molecular mechanism of PGB1 amyloid formation, we designed a set of variants with mutations that change the local secondary structure propensity in PGB1, but have similar global conformational stability. The kinetics of amyloid formation of all these variants have been studied and compared. Our results show that different locations of even a single mutation can have a dramatic effect on PGB1 amyloid formation, which is in sharp contrast with a previous report. Our results also suggest that the "-helix in PGB1 plays an important role in the amyloid formation process of PGB1. In the final study, we investigate the forces that contribute to protein stability in a very general manner. Based on what we have learned about the major forces that contribute to the stability of globular proteins, protein stability should increase as the size of the protein increases. This is not observed: the conformational stability of globular proteins is independent of protein size. In an effort to understand why large proteins are not more stable than small proteins, twenty single-domain globular proteins ranging in size from 35 to 470 residues have been analyzed. Our study shows that nature buries more charged groups and more non-hydrogen-bonded polar groups to destabilize large proteins.

Protein structure and motion plays an essential role in nearly all forms of life. Understanding both protein folding and protein conformational change can bring deeper insight to many biochemical processes and even into some devastating diseases thought to be the result of protein misfolding. Experimental methods are currently unable to capture detailed, large-scale motions. Traditional computational approaches (e.g., molecular dynamics and Monte Carlo simulations) are too expensive to simulate time periods long enough for anything but small peptide fragments. This research aims to model such molecular movement using a motion framework originally developed for robotic applications called the Probabilistic Roadmap Method. The Probabilistic Roadmap Method builds a graph, or roadmap, to model the connectivity of the movable object?s valid motion space. We previously applied this methodology to study protein folding and obtained promising results for several small proteins. Here, we extend our existing protein folding framework to handle larger proteins and to study a broader range of motion problems. We present a methodology for incrementally constructing roadmaps until they satisfy a set of evaluation criteria. We show the generality of this scheme by providing evaluation criteria for two types of motion problems: protein folding and protein transitions. Incremental Map Generation eliminates the burden of selecting a sampling density which in practice is highly sensitive to the protein under study and difficult to select. We also generalize the roadmap construction process to be biased towards multiple conformations of interest thereby allowing it to model transitions, i.e., motions between multiple known conformations, instead of just folding to a single known conformation. We provide evidence that this generalized motion framework models large-scale conformational change more realistically than competing methods. We use rigidity theory to increase the efficiency of roadmap construction by introducing a new sampling scheme and new distance metrics. It is only with these rigidity-based techniques that we were able to detect subtle folding differences between a set of structurally similar proteins. We also use it to study several problems related to protein motion including distinguishing secondary structure formation order, modeling hydrogen exchange, and folding core identification. We compare our results to both experimental data and other computational methods.

Thesis (Ph. D.)--University of Chicago, Department of Chemistry, 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 193-199). Available also in a digital version from Dissertation Abstracts.

Not available / text

The twin-arginine translocase allows the translocation of folded protein substrates across the cytoplasmic membrane of bacteria and archaea or the thylakoid membrane of plants. In Escherichia coli, its protein components TatA, TatB and TatC assemble dynamically upon interaction with protein substrates. Prior to export, the machinery performs a quality control check so that only correctly folded proteins are translocated. The first objective of this work was to derive and apply new methodologies based on the inherent qualities of the pathway. We developed a new bacterial two-hybrid system that capitalizes on the folding quality control mechanism of the Tat pathway. One protein (prey) is fused to Tat-specific signal peptide. A second (bait) protein is produced as a fusion to a reporter that produces a "signal" (growth or enzymatic activity) only when the bait-reporter fusion binds to the prey and the resulting complex is exported into the periplasm via the Tat pathway. As a second biotechnological application of the Tat pathway, we developed a phage display system that allows the protein of interest to fold within the cytoplasm prior export and display onto phage particles. This is in contrast to the conventional phage display system, in which displayed protein folds in the periplasm. We took advantage of this new system to screen a library of 2 x 10⁶ of fluorescent GFP variants containing a hexameric peptide insertion for ligand binding. Despite the diversity of the hexamer, we were not able to isolate single GFP variants that would bind with specificity to various ligands. This highlights the difficulty in engineering GFP variants that can bind to other proteins while retaining the ability to fluoresce. The second aspect of this research was to examine mechanistic aspects of the Tat pathway. TatB and TatC are responsible for the recognition of Tat signal peptides. Here, we established the importance of TatC as the crucial component of the Tat pathway for the interaction with the hallmark twin-arginine motif within Tat signal peptide. Substitution of the RR dipeptide with a KK sequence completely abolishes export. In a genetic screen using a ssTorA(KK)-GFP-SsrA as a reporter. We identified several amino acid substitutions within TatC that allowed the alteration of the substrate specificity of the pathway as indicated by the impairment of indigenous Tat substrates. Finally, we analyzed the conformational dynamics of TatA using GFP fusions and by incorporation of the chemically reactive, non-canonical amino acid azidohomoalanine.

High-speed and low resolution analog-to-digital converters (ADC) are key elements in the read channel of optical and magnetic data storage systems. The required resolution is about 6-7 bits while the sampling rate and effective resolution bandwidth requirements increase with each generation of storage system. Folding is a technique to reduce the number of comparators used in the flash architecture. By means of an analog preprocessing circuit in folding A/D converters the number of comparators can be reduced significantly. Folding architectures exhibit low power and low latency as well as the ability to run at high sampling rates. Folding ADCs employing interpolation schemes to generate extra folding waveforms are called "Folding and Interpolating ADC" (F&I ADC). The aim of this research is to increase the input bandwidth of high speed conversion, and low latency F&I ADC. Behavioral models are developed to analyze the bandwidth limitation at the architecture level. A front-end sample-and-hold unit is employed to tackle the frequency multiplication problem, which is intrinsic for all F&I ADCs. Current-mode signal processing is adopted to increase the bandwidth of the folding amplifiers and interpolators, which are the bottleneck of the whole system. An operational transconductance amplifier (OTA) based folding amplifier, current mirror-based interpolator, very low impedance fast current comparator are proposed and designed to carry out the current-mode signal processing. A new bit synchronization scheme is proposed to correct the error caused by the delay difference between the coarse and fine channels. A prototype chip was designed and fabricated in 0.35μm CMOS process to verify the ideas. The S/H and F&I ADC prototype is realized in 0.35μm double-poly CMOS process (only one poly is used). Integral nonlinearity (INL) is 1.0 LSB and Differential nonlinearity (DNL) is 0.6 LSB at 110 KHz. The ADC occupies 1.2mm2 active area and dissipates 200mW (excluding 70mW of S/H) from 3.3V supply. At 300MSPS sampling rate, the ADC achieves no less than 6 ENOB with input signal lower than 60MHz. It has the highest input bandwidth of 60MHz reported in the literature for this type of CMOS ADC with similar resolution and sample rate.