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Described in this thesis is the use of controlled radical polymerization techniques to synthesize tetrazine end-functionalized telechelic polymers, which were employed to generate T4 Lysozyme homodimers. A mutant of T4 Lysozyme (V131C), containing a single surface-exposed cysteine residue, was modified via Michael addition with a protein-reactive trans-cyclooctene. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to obtain telechelic poly(N-isopropylacrylamide) (pNIPAAm) polymers with Mn (1H-NMR) of 2.0 kDa and molecular weight dispersity, Đ, (GPC) of 1.05. pNIPAAm was modified at both ends with (4-(6- methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanol. As a control, a 2.0 kDa bis-carboxylic acid poly(ethylene glycol) was modified at both ends with (4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl)methanol and a 2.0 kDa bis-maleimide pNIPAAm was also synthesized. Tetrazine- functionalized polymers were then ligated to a trans-cyclooctene modified Lysozyme (T4L-TCO). Ligation of T4L-TCO to bis-tetrazine pNIPAAm resulted in 27.27% yield of the homodimeric conjugate, whereas bis-tetrazine PEG resulted in 16.75% and bis-maleimide pNIPAAm resulted in <1% yield after 1 hour with no significant increase in yield during extended incubation times up to 24 hours. This work shows that tetrazine chemistry results in a higher yield of dimeric protein conjugation for both PEG and CRP polymers.

Described in this thesis is the use of controlled radical polymerization techniques to synthesize tetrazine end-functionalized telechelic polymers, which were employed to generate T4 Lysozyme homodimers. A mutant of T4 Lysozyme (V131C), containing a single surface-exposed cysteine residue, was modified via Michael addition with a protein-reactive trans-cyclooctene. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to obtain telechelic poly(N-isopropylacrylamide) (pNIPAAm) polymers with Mn (1H-NMR) of 2.0 kDa and molecular weight dispersity, Đ, (GPC) of 1.05. pNIPAAm was modified at both ends with (4-(6- methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanol. As a control, a 2.0 kDa bis-carboxylic acid poly(ethylene glycol) was modified at both ends with (4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl)methanol and a 2.0 kDa bis-maleimide pNIPAAm was also synthesized. Tetrazine- functionalized polymers were then ligated to a trans-cyclooctene modified Lysozyme (T4L-TCO). Ligation of T4L-TCO to bis-tetrazine pNIPAAm resulted in 27.27% yield of the homodimeric conjugate, whereas bis-tetrazine PEG resulted in 16.75% and bis-maleimide pNIPAAm resulted in <1% yield after 1 hour with no significant increase in yield during extended incubation times up to 24 hours. This work shows that tetrazine chemistry results in a higher yield of dimeric protein conjugation for both PEG and CRP polymers.

Conformational change in protein-ligand complexes is widely modeled, but the protein accommodation expected on binding a congeneric series of ligands has received less attention. Given their use in medicinal chemistry, there are surprisingly few substantial series of congeneric ligand complexes in the Protein Data Bank (PDB). Here we determine the structures of eight alkyl benzenes, in single-methylene increases from benzene to n-hexylbenzene, bound to an enclosed cavity in T4 lysozyme. The volume of the apo cavity suffices to accommodate benzene but, even with toluene, larger cavity conformations become observable in the electron density, and over the series two other major conformations are observed. These involve discrete changes in main-chain conformation, expanding the site; few continuous changes in the site are observed. In most structures, two discrete protein conformations are observed simultaneously, and energetic considerations suggest that these conformations are low in energy relative to the ground state. An analysis of 121 lysozyme cavity structures in the PDB finds that these three conformations dominate the previously determined structures, largely modeled in a single conformation. An investigation of the few congeneric series in the PDB suggests that discrete changes are common adaptations to a series of growing ligands. The discrete, but relatively few, conformational states observed here, and their energetic accessibility, may have implications for anticipating protein conformational change in ligand design.

The structure of the unusually long (~100 amino-acid residues) N-terminal domain of the light-harvesting protein CP29 of plants is not defined in the crystal structure of this membrane protein. We studied the N-terminus using two electron paramagnetic resonance (EPR) approaches: the rotational diffusion of spin labels at 55 residues with continuous-wave EPR, and three sets of distances with a pulsed EPR method. The N-terminus is relatively structured. Five regions that differ considerably in their dynamics are identified. Two regions have low rotational diffusion, one of which shows a-helical character suggesting contact with the protein surface. This immobile part is flanked by two highly dynamic, unstructured regions (loops) that cover residues 10-22 and 82-91. These loops may be important for the interaction with other light-harvesting proteins. The region around residue 4 also has low rotational diffusion, presumably because it attaches noncovalently to the protein. This section is close to a phosphorylation site (Thr-6) in related proteins, such as those encoded by the Lhcb4.2 gene. Phosphorylation might influence the interaction with other antenna complexes, thereby regulating the supramolecular organization in the thylakoid membrane.

Proteins in solution exhibit structural fluctuations on a wide range of characteristic time scales, from fast backbone motions in the picosecond to nanosecond range to slow conformational exchange in the microsecond to millisecond time domain. These motions can play important roles in function, thus elucidation of molecular mechanisms underlying function requires experimental techniques capable of measuring motions on these time scales. Site directed spin labeling and EPR spectroscopy (SDSL-EPR) has been established as an important tool in protein science for studying structure, identifying dynamically disordered sequences, and monitoring conformational switching triggered by chemical or physical signals in soluble and membrane-bound proteins. However, it remains to be investigated whether fast backbone motions contribute to the spectra of the spin label side chain R1 in well-ordered sequences of regular secondary structure, and how slow conformational exchange between substates can be detected at equilibrium; these are goals of this dissertation. The results from this work support a model wherein variations in the EPR spectral lineshape of R1-labeled proteins measures the amplitude of fast but constrained backbone fluctuations on the nanosecond time scale, and show that these motions are correlated with the local packing density. In addition, it is shown that osmolyte perturbation SDSL (OP-SDSL) can be used to identify conformational equilibria between substates that have characteristic lifetimes > 100 ns. The newly-developed OP-SDSL strategy was employed along with continuous wave (CW) lineshape analysis to map molecular flexibility of sperm whale myoglobin in folded and partially folded states and of core-repacking mutants of T4 lysozyme. For myoglobin, the results reproduce and extend earlier NMR studies, thus validating the OP-SDSL method and highlighting advantages of the inherent EPR time scale. For T4 lysozyme, it is found that core repacking mutations th

Proteins in solution exhibit structural fluctuations on a wide range of characteristic time scales, from fast backbone motions in the picosecond to nanosecond range to slow conformational exchange in the microsecond to millisecond time domain. These motions can play important roles in function, thus elucidation of molecular mechanisms underlying function requires experimental techniques capable of measuring motions on these time scales. Site directed spin labeling and EPR spectroscopy (SDSL-EPR) has been established as an important tool in protein science for studying structure, identifying dynamically disordered sequences, and monitoring conformational switching triggered by chemical or physical signals in soluble and membrane-bound proteins. However, it remains to be investigated whether fast backbone motions contribute to the spectra of the spin label side chain R1 in well-ordered sequences of regular secondary structure, and how slow conformational exchange between substates can be detected at equilibrium; these are goals of this dissertation. The results from this work support a model wherein variations in the EPR spectral lineshape of R1-labeled proteins measures the amplitude of fast but constrained backbone fluctuations on the nanosecond time scale, and show that these motions are correlated with the local packing density. In addition, it is shown that osmolyte perturbation SDSL (OP-SDSL) can be used to identify conformational equilibria between substates that have characteristic lifetimes > 100 ns. The newly-developed OP-SDSL strategy was employed along with continuous wave (CW) lineshape analysis to map molecular flexibility of sperm whale myoglobin in folded and partially folded states and of core-repacking mutants of T4 lysozyme. For myoglobin, the results reproduce and extend earlier NMR studies, thus validating the OP-SDSL method and highlighting advantages of the inherent EPR time scale. For T4 lysozyme, it is found that core repacking mutations th

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments are extremely powerful for characterizing millisecond time-scale conformational exchange processes in biomolecules. A large number of such CPMG experiments have now emerged for measuring protein backbone chemical shifts of sparsely populated (> 0.5%), excited state conformers that cannot be directly detected in NMR spectra and that are invisible to most other biophysical methods as well. A notable deficiency is, however, the absence of CPMG experiments for measurement of H-1(alpha) and C-13(alpha) chemical shifts of glycine residues in the excited state that reflects the fact that in this case the H-1(alpha), C-13(alpha) spins form a three-spin system that is more complex than the AX H-1(alpha)-C-13(alpha) spin systems in the other amino acids. Here pulse sequences for recording H-1(alpha) and C-13(alpha) CPMG relaxation dispersion profiles derived from glycine residues are presented that provide information from which H-1(alpha), C-13(alpha) chemical shifts can be obtained. The utility of these experiments is demonstrated by an application to a mutant of T4 lysozyme that undergoes a millisecond time-scale exchange process facilitating the binding of hydrophobic ligands to an internal cavity in the protein.

Crystal structures of an inactive mutant (D308A) of the lytic transglycosylase MltA from Escherichia coli have been determined in two different apo-forms, as well as in complex with the substrate analogue chitohexaose. The chitohexaose binds with all six saccharide residues in the active site groove, with an intact glycosidic bond at the bond cleavage center. Its binding induces a large reorientation of the two structural domains in MltA, narrowing the active site groove and allowing tight interactions of the oligosaccharide with residues from both domains. The structures identify residues in MltA with key roles in the binding and recognition of peptidoglycan and confirm that Asp-308 is the single catalytic residue, acting as a general acid/base. Moreover, the structures suggest that catalysis involves a high energy conformation of the scissile glycosidic linkage and that the putative oxocarbenium ion intermediate is stabilized by the dipole moment of a nearby alpha-helix.

To investigate the structural and thermodynamic basis of the binding of solvent at internal sites within proteins a number of mutations were constructed in T4 lysozyme. Some of these were designed to introduce new solvent-binding sites. Others were intended to displace solvent from preexisting sites. In one case Val-149 was replaced with alanine, serine, cysteine, threonine, isoleucine, and glycine. Crystallographic analysis shows that, with the exception of isoleucine, each of these substitutions results in the binding of solvent at a polar site that is sterically blocked in the wild-type enzyme. Mutations designed to perturb or displace a solvent molecule present in the native enzyme included the replacement of Thr-152 with alanine, serine, cysteine, valine, and isoleucine. Although the solvent molecule was moved in some cases by up to 1.7 A, in no case was it completely removed from the folded protein. The results suggest that hydrogen bonds from the protein to bound solvent are energy neutral. The binding of solvent to internal sites within proteins also appears to be energy neutral except insofar as the bound solvent may prevent a loss of energy due to potential hydrogen bonding groups that would otherwise be unsatisfied. The introduction of a solvent-binding site appears to require not only a cavity to accommodate the water molecule but also the presence of polar groups to help satisfy its hydrogen-bonding potential. It may be easier to design a site to accommodate two or more water molecules rather than one as the solvent molecules can then hydrogen-bond to each other. For similar reasons it is often difficult to design a point mutation that will displace a single solvent molecule from the core of a protein.

To investigate the structural and thermodynamic basis of the binding of solvent at internal sites within proteins a number of mutations were constructed in T4 lysozyme. Some of these were designed to introduce new solvent-binding sites. Others were intended to displace solvent from preexisting sites. In one case Val-149 was replaced with alanine, serine, cysteine, threonine, isoleucine, and glycine. Crystallographic analysis shows that, with the exception of isoleucine, each of these substitutions results in the binding of solvent at a polar site that is sterically blocked in the wild-type enzyme. Mutations designed to perturb or displace a solvent molecule present in the native enzyme included the replacement of Thr-152 with alanine, serine, cysteine, valine, and isoleucine. Although the solvent molecule was moved in some cases by up to 1.7 A, in no case was it completely removed from the folded protein. The results suggest that hydrogen bonds from the protein to bound solvent are energy neutral. The binding of solvent to internal sites within proteins also appears to be energy neutral except insofar as the bound solvent may prevent a loss of energy due to potential hydrogen bonding groups that would otherwise be unsatisfied. The introduction of a solvent-binding site appears to require not only a cavity to accommodate the water molecule but also the presence of polar groups to help satisfy its hydrogen-bonding potential. It may be easier to design a site to accommodate two or more water molecules rather than one as the solvent molecules can then hydrogen-bond to each other. For similar reasons it is often difficult to design a point mutation that will displace a single solvent molecule from the core of a protein.

Several variants of T4 lysozyme have been identified that sequester small organic ligands in cavities or clefts. To evaluate potential binding sites for non-polar molecules, we screened a number of hydrophobic large-to-small mutants for stabilization in the presence of benzene. In addition to Leu99-->Ala, binding was indicated for at least five other mutants. Variants Met102-->Ala and Leu133-->Gly, and a crevice mutant, Phe104-->Ala, were further characterized using X-ray crystallography and thermal denaturation. As predicted from the shape of the cavity in the benzene complex, mutant Leu133-->Gly also bound p-xylene. We attempted to enlarge the cavity of the Met102-->Ala mutant into a deep crevice through an additional substitution, but the double mutant failed to bind ligands because an adjacent helix rearranged into a non-helical structure, apparently due to the loss of packing interactions. In general, the protein structure contracted slightly to reduce the volume of the void created by truncating substitutions and expanded upon binding the non-polar ligand, with shifts similar to those resulting from the mutations.A polar molecule binding site was also created by truncating Arg95 to alanine. This creates a highly complementary buried polar environment that can be utilized as a specific "receptor" for a guanidinium ion. Our results suggest that creating a deficiency through truncating mutations of buried residues generates "binding potential" for ligands with characteristics similar to the deleted side-chain. Analysis of complex and apo crystal structures of binding and non-binding mutants suggests that ligand size and shape as well as protein flexibility and complementarity are all determinants of binding. Binding at non-polar sites is governed by hydrophobicity and steric interactions and is relatively permissive. Binding at a polar site is more restrictive and requires extensive complementarity between the ligand and the site.

To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non-polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the "large-to-small" substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts < or = 0.3 A); in other cases mainchain atoms moved up to 2 A. In the case of Ile 29 --> Ala the structure collapsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 --> Ala (V149A) and Met 6 --> Ala (M6A), however, are exceptions and have, respectively, one and two well-ordered water molecules within the cavity. The Val 149 --> Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild-type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 --> Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., -2.1 kcal/mol for all Leu --> Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol(-1) A(-3). Physically, this term is due to the loss of van der Wa

Several variants of T4 lysozyme have been identified that sequester small organic ligands in cavities or clefts. To evaluate potential binding sites for non-polar molecules, we screened a number of hydrophobic large-to-small mutants for stabilization in the presence of benzene. In addition to Leu99-->Ala, binding was indicated for at least five other mutants. Variants Met102-->Ala and Leu133-->Gly, and a crevice mutant, Phe104-->Ala, were further characterized using X-ray crystallography and thermal denaturation. As predicted from the shape of the cavity in the benzene complex, mutant Leu133-->Gly also bound p-xylene. We attempted to enlarge the cavity of the Met102-->Ala mutant into a deep crevice through an additional substitution, but the double mutant failed to bind ligands because an adjacent helix rearranged into a non-helical structure, apparently due to the loss of packing interactions. In general, the protein structure contracted slightly to reduce the volume of the void created by truncating substitutions and expanded upon binding the non-polar ligand, with shifts similar to those resulting from the mutations.A polar molecule binding site was also created by truncating Arg95 to alanine. This creates a highly complementary buried polar environment that can be utilized as a specific "receptor" for a guanidinium ion. Our results suggest that creating a deficiency through truncating mutations of buried residues generates "binding potential" for ligands with characteristics similar to the deleted side-chain. Analysis of complex and apo crystal structures of binding and non-binding mutants suggests that ligand size and shape as well as protein flexibility and complementarity are all determinants of binding. Binding at non-polar sites is governed by hydrophobicity and steric interactions and is relatively permissive. Binding at a polar site is more restrictive and requires extensive complementarity between the ligand and the site.

To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non-polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the "large-to-small" substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts < or = 0.3 A); in other cases mainchain atoms moved up to 2 A. In the case of Ile 29 --> Ala the structure collapsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 --> Ala (V149A) and Met 6 --> Ala (M6A), however, are exceptions and have, respectively, one and two well-ordered water molecules within the cavity. The Val 149 --> Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild-type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 --> Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., -2.1 kcal/mol for all Leu --> Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol(-1) A(-3). Physically, this term is due to the loss of van der Wa