In the present work I have developed and characterized an in vivo selection system to investigate molecular evolution. The essential gene that codes for adenylate kinase (AK; EC 2.4.7.3) in the gram-positive moderate thermophile Geobacillus stearothermophilus was replaced through homologous recombination with its counterpart from the mesophilic Bacillus subtilis. PCR, DNA sequencing and Southern analysis confirmed proper gene replacement and preservation of neighboring genes in the recombinant strain. Recombinant cells (NUB3621-R:ThEV) displayed a temperature sensitive phenotype, with a highest growing temperature almost 20°C lower than that of wild-type cells (56°C vs 75°C). The temperature sensitive phenotype in recombinant cells was linked to a disruption of adenylate homeostasis at high temperatures, secondary to B. subtilis AK heat inactivation, as shown by enzyme activity assays, temperature denaturation profiles and adenylate level measurements. Evolution of a single gene, B. subtilis adk, was investigated by steady-state growth of the recombinant strain from 55 to 70°C in a turbidostat. The temporal characteristics of B. subtilis adk evolution were probed by DNA sequence analysis at various temperatures. The appearance of more fit strains from pre-existing genetic variation, rapid extinctions generated by clonal interference and selection coupled to a strong dependence on historical context and chance events resulted in a punctuated pattern of evolution. The organism fitness at different temperatures could be linked to its expressed AK variant and allowed the investigation of the biochemical and structural basis of adaptation. Heat denaturation and enzyme activity studies showed that all isolated AK mutants were more stable than the wild-type protein and were responsible for the punctuated pattern of evolution detected during the population analyses. The atomic structure of one of the isolated AK variants (Q199R) revealed that its increased thermostability is due to unique electrostatic interactions absent in the wild-type structure. Taken together our population, biochemical and structural analysis suggests that natural selection observed at the molecular level is guided by the same principles that act at the organismal level.

The mechanical properties of the outer hair cells (OHCs) lateral wall are essential to elucidate the mechanism of OHC electromotility. Optical tweezers were used to characterize the mechanical properties of OHC plasma membrane (PM) by pulling tethers. A greater force was required for tether formations from OHC lateral wall (499 pN +/- 152) than from OHC basal end (142 pN +/- 49), consistent with the presence of a more extensive cytoskeleton support beneath the PM at the lateral wall. The apparent PM stiffness, which was estimated by measuring tether force at different tether length, was 3.71 pN/mum for OHC lateral wall and 4.57 pN/mum for OHC basal end. The apparent PM viscosity was measured by pulling tethers at different rates while continuously recording the tether force, and was estimated in the range of 13--33 pN·s/mum.

The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 A by multiwavelength anomalous dispersion phasing methods using selenomethionyl GFP crystals. The protein is in the shape of a cylinder, comprising 11 strands of $\beta$-sheet with an $\alpha$-helix inside and short helical segments on the ends of the cylinder. This motif, with $\beta$-structure on the outside and $\alpha$-helix on the inside, represents a new protein fold. Two protomers pack closely together to form a dimer in the crystal. The fluorophores are protected inside the cylinders, and their structures are consistent with the formation of aromatic systems made up of Tyr66 with oxidation of its C$\sb{\alpha}$-C$\sb{\beta}$ bond coupled with cyclization of the neighboring glycine and serine residues. The environment inside the cylinder explains the effects of many existing mutants of GFP and suggests which side chains could be modified to change the spectral properties of GFP. Furthermore, the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein. GFP can be reduced by sodium dithionite and as a result, loses its fluorescence. The structure of reduced GFP has been solved which shows that the side chain of Tyr66 at the fluorophore is the group being reduced. While oxidized, the C$\sb{\alpha}$ atom of Tyr66 is not a chiral center since all fluorophore atoms are co-planar. After reduction, the C$\sb{\alpha}$ atom returns to a chiral center, but has either an L or D configuration, indicating that the breakage of the resonance system eliminates the fluorescence of GFP and the reduction of Tyr66 is not stereospecific.

The range of conformations of macromolecules and the dynamic interconversion between conformations is an important part of the relationship between structure and function. The existence of side-chain conformational substates in two systems is directly demonstrated using traditional X-ray crystallographic refinement methodologies. One system is a mutant met-myoglobin where the phenylalanine at position 46 was replaced by a valine. The other is a low-temperature high-resolution dataset for wild-type CO myoglobin, Multi-conformer refinements, which combine molecular dynamics with X-ray data restraints, are shown to model side-chain substates similar to those identified manually. The two conformations of his64 in the mutant myoglobin and wild-type myoglobin, and three conformations of ser117 in the wild-type myoglobin are found "automatically." Time averaged refinements, which also use a modified molecular dynamics algorithm but is a simulation rather than strictly an optimization, also found similar side-chain conformations. The time averaged refinement for the mutant myoglobin found 5 transitions of the distal histidine in only 30 nominal ps of simulation time. The singular value decomposition (SVD), when coupled with the coefficient of kurtosis for the first right singular vector, is a simple but powerful "filter" for identifying bi-conformer side-chains from large ensembles of structures. Those residues identified with the SVD as having two discrete substates from the time-averaged refinement agree fairly closely with those found manually. The SVD has also proven to be a powerful tool for analyzing conformational substates for the protein as a whole. The configuration space projection of a Ins solvated myoglobin MD simulation using the SVD shows a "beads on a string" motif, suggesting a hierarchical topology reminiscent of substates. Comparing the left singular vectors from two halves of a dynamics simulation shows that even when only C a atoms are used, the configuration spaces searched by the solvated Mb simulation do not match. This quantifies what can already be seen qualitatively in the configuration space portraits of the system. When time-averaged refinement trajectories are compared, there is a much higher degree of similarity indicating that the accelerated dynamics system is approaching ergodicity.

The first stage in hemostasis and thrombosis is the binding of the platelet membrane receptor, glycoprotein (GP) Ib-IX, to its ligand, von Willebrand factor (VWF), in the subendothelium. Using an optical tweezers system, we have measured the binding strength of: (1) VWF variants (unusually large VWF (ULVWF), plasma VWF, and isolated VWF A1 domain) to GP Ib-IX receptors; (2) GP Ib-IX variants (receptors with human-canine chimeric sequences in the leucine-rich domain, and truncated receptors) to plasma VWF; (3) plasma VWF to gain-of-function (GOF) and loss-of-function (LOF) mutated GP Ib-IX receptors; (4) VWF Al domain to clustered GP Ib-IX receptors; and (5) ULVWF multimers to GP Ib-IX in the presence of the VWF cleaving protease (ADAMTS-13) and other modulators. In addition, we dynamically measured unbinding force profiles between A1 and GP Ib-IX at loading rates ranging from 200--20,000 pN/s and examined the relationship between bond rupture force and loading rate. In the absence of shear stress, ULVWF multimers formed spontaneous high-strength bonds with GP Ib-IX, while plasma VWF required exogenous modulators. The strength of individual bonds formed with GP Ib-IX was similar for both ULVWF and the isolated A1-domain and greater than those of plasma VWF induced by an exogenous modulator. We observed ULVWF/GP Ib-IX binding was completely abolished with the addition of either ADAMTS-13 or plasma cryosupernatant. We found that the putative single bond strengths between A1 and GP Ibalpha GOF mutants were significantly greater than the A1/wild-type GP Ib-IX bond at all loading rates examined. In addition, the GP Ibalpha LOF mutants exhibited significantly lower putative single bond strengths with A1 than the wild-type receptors. The experiments involving the adhesion of either chimeric or truncated GP Ib-IX receptors to VWF illustrated the importance of various regions of the glycoprotein in properly binding VWF. The minimal detachment force between GP Ib-IX and A1 or plasma VWF doubled after an exogenous clustering agent was added, thereby demonstrating the importance of avidity, as opposed to affinity, modulation. Finally, we observed a linear relationship between the rupture force and the logarithm of the loading rate, consistent with the Bell Model.

The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis. The DNA replication process in RB69 (a T4-like bacteriophage) is similar to the processes in eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome. In order to examine protein-protein interactions at the DNA replication fork, solution conditions have been established for the formation of a discrete and homogeneous complex of RB69 DNA polymerase (gp43), primer-template DNA and RB69 single-stranded DNA binding protein (gp32) using equilibrium fluorescence and light scattering. The interaction between DNA polymerase and single-stranded DNA binding protein has been characterized by fluorescence titrations and results in a 60-fold increase in the overall affinity of RB69 SSB for template-strand DNA in the presence of DNA polymerase. Our data further suggest that the cooperative binding of the RB69 DNA polymerase and SSB to the primer-template junction is a simple but functionally important means of regulatory assembly of replication proteins at the site-of-action. A functional domain of RB69 single-stranded DNA-binding protein previously suggested to be the site of RB69 DNA polymerase:SSB interactions has been shown to be dispensable. The data from these studies have been used to model the RB69 DNA polymerase:SSB interaction at the primer-template junction. Fusion of RB69 SSB with its cognate DNA polymerase via a short six amino acid linker increases affinity for primer-template DNA by 6-fold and increases processivity by 7-fold while maintaining fidelity. The crystal structure of this fusion protein was solved by a combination of multiwavelength anomalous diffraction and molecular replacement to 3.2 A resolution and shows that RB69 SSB is positioned proximal to the N-terminal domain of RB69 DNA polymerase near the template strand entry channel. The structural and biochemical data suggest that SSB interactions with DNA polymerase are transient and flexible, consistent with models of a dynamic replisome during elongation.

The material properties of biomembranes can be measured by forming a tether, a thin bilayer tube that extends from the membrane surface. The force required to maintain a tether at a given length depends upon both membrane properties as well as the mechanical, chemical and electrical environment. To characterize membrane material properties and responses, the influences of electromechanical energy, interfacial phenomena, and thermally-driven entropic tensions are considered in separate, thermodynamic models of tether formation. To determine how electric fields influence tether behavior, the energetic contributions arising from Maxwell stresses as well as from flexoelectric and piezoelectric coupling are included in an analysis of tether formation from an aspirated vesicle. For typical membrane elctromechanical coefficients, flexoelectric coupling alters the force required to form a tether of a given length, while piezoelectric coupling and Maxwell forces do not greatly change the force vs. tether length behavior. Given recent experiments demonstrate tethers formed from cellular membranes are sensitive to the transmembrane potential, an analysis is developed to characterize the electromechanical properties of unaspirated cellular membranes. Both flexoelectric and piezoelectric coupling energies as well as the voltage sensitivity of the interfacial tension and bending stiffness are included in the analysis. For typical membrane charge densities, small changes in tether force are calculated for the contributions of the interfacial phenomena. Inclusion of the electromechanical coupling energies leads to experimentally observable changes in tether force. These analyses are applied to determine the mechanism by which the motor protein prestin confers electromotility to the outer hair cell (OHC) of the mammalian cochlea. For the values of electromechanical coupling coefficients obtained from OHC deformation models, the tether force increases with depolarization for flexoelectric coupling and decreases for piezoelectric coupling. Since cellular membranes are typically under small entropically-driven tensions, the influence of thermally driven surface undulations on tether conformation is considered. By fitting the model to experimental tether data, the tension of a vesicle can be determined. The analyses developed in this thesis provide novel methods to determine many otherwise difficult to characterize membrane material properties and, thus, help to deepen understanding of the behavior of cellular membranes.

Subtle differences in Fe-C-O geometry between the recombinant (pH 9, space group P6) and native (pH 7, space group P21) crystal forms of carbon monoxide sperm whale myoglobin are magnified by using n-butyl isocyanide as a probe of distal pocket stereochemistry. Crystal structures of methyl, ethyl, n-propyl and n-butyl isocyanide bound to recombinant wild-type myoglobin were determined and then compared with the corresponding native myoglobin isocyanide structures determined by Ken Johnson (Ph.D. Dissertation, Rice University, 1993). The wild-type structures show all the ligands pointing inward and the distal histidine in the "down" conformation, whereas the native myoglobin n-propyl and n-butyl isocyanide structures show the ligand side chain pointing outward causing upward movement of the distal histidine. In order to examine the causes of these differences, the structures of Mb n-butyl isocyanide in native P21 crystals and recombinant V68F P6 crystals were determined at both pH 7 and 9. The results show that pH and not crystal packing is the key determinant of ligand orientation. Neutral or low pH favors rotation of the distal histidine and outward movement of the side chain of bound n-butyl isocyanide. Four mutant Mb structures were determined to examine the roles of the distal histidine and valine residues in governing ligand orientation. The results demonstrate that His64 (E7) sterically restricts outward movement of the isocyanide side chain since its replacement with alanine (H64A) or an increase in its mobility (F46V) causes the appearance of the "out" ligand conformation. Steric crowding in the protein interior by the V68F mutation is not sufficient to cause the ligand to point outward at pH 9.0. Since the rate and equilibrium constants for myoglobin n-butyl isocyanide show only a small pH dependence, the "in" and "out" conformations appear to have similar free energies. This suggests that ligand movement into the protein interior and outward through the distal histidine gate have similar probabilities which is what has been observed in laser photolysis experiments with oxymyoglobin (Scott, E. E. and Gibson, Q. H. (1997) Biochemistry 36, 11909--11917).

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is a prototype for the family of eukaryotic RNA processing proteins containing the common RNA recognition motif (RRM). The region consisting of residues 1--195 of hnRNP Al is referred to as UP1. This region has two RRMs and has a high affinity for both single-stranded RNA and the human telomeric repeat sequence d(TTAGGG)n. We have used UP1's novel DNA binding to investigate how RRMs bind nucleic acid bases through their highly conserved RNP consensus sequences. Nine complexes of UP1 bound to modified telomeric repeats were investigated using equilibrium fluorescence binding and X-ray crystallography. In two of the complexes, alteration of a guanine to either 2-aminopurine or nebularine resulted in an increase in Kd from 70 nM to 160 and 280 nM, respectively. The loss of orienting interactions between UP1 and the substituted base allows it to flip between syn and anti conformations. Substitution of the same base with 7-deaza-guanine preserves the O6/N1 contacts but still increases the Kd to 250 nM, a result suggesting that it is not simply the loss of affinity that gives rise to base mobility but also the stereochemistry of the specific contact to O6. Although these studies provide details of UP1 interactions to nucleic acids, three general observations about RRMs are also evident: (1) as suggested by informatic studies, main chain to base hydrogen bonding make up an important aspect of ligand recognition; (2) steric clashes generated by modification of a hydrogen bond donor-acceptor pair to a donor-donor pair are poorly tolerated; and (3) a conserved lysine position proximal to RNP-2 (K106-IFVGGI) orients the purine to allow stereochemical discrimination between adenine and guanine based on the 6-position. This single interaction is well-conserved in known RRM structures and appears to be a broad indicator for purine preference in the larger family of RRM proteins.

Hydrophobic matching, in which transmembrane proteins cause the surrounding lipid bilayer to adjust its thickness to match the hydrophobic surface of the protein, is a commonly accepted idea in biophysics, but one that until now has not been experimentally tested. One important consequence is that protein interactions will be mediated by the energy cost of deforming the membrane from its protein free state. With X-ray scattering techniques we tested these ideas with the peptide gramicidin embedded in DLPC and DMPC bilayers. Gramicidin pushes the different membranes to a common thickness as expected from hydrophobic matching. Concurrently, gramicidin-gramicidin nearest neighbor distance decreases with increasing mismatch, which confirms that the strain in the lipid bilayer gives rise to an attractive potential between the proteins. We have taken a continuum theory approach to the analysis of the experimental results. This approach treats the energetics of membrane-protein interactions as a function of the material properties of the membrane such as bending rigidity and compressibility. Using numerical methods and a novel simulation technique, we have successfully demonstrated the theoretical relationship between membrane thickness change and protein correlation. By quantitatively reproducing our experimental results, we have shown that the theory of membrane deformation is sufficient to explain the phenomena of hydrophobic matching. We also include a study on the peptide melittin as an example of the type of protein-lipid system we want to understand better. We answer the question of the orientation of the peptide when making membrane pores.