R-3 Repository :: Browsing by Author "Phillips, George N., Jr."

Browsing by Author "Phillips, George N., Jr."

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A new global optimization strategy for the molecular replacement problem
(2002) Jamrog, Diane Christine; Zhang, Yin; Phillips, George N., Jr.; Tapia, Richard A.
The primary technique for determining the three-dimensional structure of a protein is X-ray crystallography, in which the molecular replacement (MR) problem arises as a critical step. Knowledge of protein structures is extremely useful for medical research, including discovering the molecular basis of disease and designing pharmaceutical drugs. This thesis proposes a new strategy to solve the MR problem, which is a global optimization problem to find the optimal orientation and position of a structurally similar model protein that will produce calculated intensities closest to those observed from an X-ray crystallography experiment. Improving the applicability and the robustness of MR methods is an important research goal because commonly used traditional MR methods, though often successful, have difficulty solving certain classes of MR problems. Moreover, the use of MR methods is only expected to increase as more structures are deposited into the Protein Data Bank. The new strategy has two major components: a six-dimensional global search and multi-start local optimization. The global search uses a low-frequency surrogate objective function and samples a coarse grid to identify good starting points for multi-start local optimization, which uses a more accurate objective function. As a result, the global search is relatively quick and the local optimization efforts are focused on promising regions of the MR variable space where solutions are likely to exist, in contrast to the traditional search strategy that exhaustively samples a uniformly fine grid of the variable space. In addition, the new strategy is deterministic, in contrast to stochastic search methods that randomly sample the variable space. This dissertation introduces a new MR program called SOMoRe that implements the new global optimization strategy. When tested on seven problems, SOMoRe was able to straightforwardly solve every test problem, including three problems that could not be directly solved by traditional MR programs. Moreover, SOMoRe also solved a MR problem using a less complete model than those required by two traditional programs and a stochastic 6D program. Based on these results, this new strategy promises to extend the applicability and robustness of MR.
Correlated atomic displacements in crystals of yeast initiatortRNA and aspartate aminotransferase analyzed by x-ray diffuse scattering
(1995) Kolatkar, Anand Ratnakar; Phillips, George N., Jr.
Biological macromolecules do not function as static molecules. A variety of biological functions are the result of fluctuations in protein and nucleic acid structure. The method of X-ray diffuse scattering analysis has been extended to provide information about intermolecular and intramolecular disorder in protein and nucleic acid crystals. Yeast initiator transfer ribonucleic acid (tRNA) carries the initial methionine to the ribosome during translation initiation. Results from analysis of the yeast initiator tRNA crystal diffuse scattering indicate that anisotropic, lattice-coupled motions contribute to the overall disorder in the crystals. The lattice-coupled disorder implies a flexing motion between the anti-codon and acceptor arms. This type of flexing has been implicated in tRNA's role in protein synthesis on the ribosome. Diffuse scattering analysis also shows that the distal half of the anti-codon arm of tRNA undergoes isotropic short-range motion correlated over a distance of approximately 3 A. This distance corresponds well with the base-pair stacking distance in RNA helices. These results are consistent with nearest-neighbor base pairs moving isotropically and as coherent units in the tRNA anti-codon arm. Aspartate aminotransferase (AspAT) is an important enzyme in the catabolism of amino acids and catalyzes the removal of their amino groups. Crystallographic evidence suggests that the small domain of one of the subunits in the biologically active dimer of AspAT in acetate buffer is disordered. Analysis of the X-ray diffuse scattering from AspAT crystals shows that the small domain moves isotropically and as a rigid body with an amplitude consistent with the crystallographically determined average B-value for the small domain. A preliminary attempt has been made to map the three-dimensional diffuse scattering from a set of simulated still diffraction photographs. This technique has been applied to actual diffraction data from AspAT crystals. The results show that an important diffuse scattering feature is weak but discernible in the reconstructed 3-D volume. X-ray diffuse scattering analysis provides unique experimental evidence for motions in biological macromolecules in crystals. Information obtained about global as well as local intramolecular motion is useful in understanding the role of molecular flexibility in biological function.
CrysFormer: Protein structure determination via Patterson maps, deep learning, and partial structure attention
(AIP Publishing LLC, 2024) Pan, Tom; Dun, Chen; Jin, Shikai; Miller, Mitchell D.; Kyrillidis, Anastasios; Phillips, George N., Jr.
Determining the atomic-level structure of a protein has been a decades-long challenge. However, recent advances in transformers and related neural network architectures have enabled researchers to significantly improve solutions to this problem. These methods use large datasets of sequence information and corresponding known protein template structures, if available. Yet, such methods only focus on sequence information. Other available prior knowledge could also be utilized, such as constructs derived from x-ray crystallography experiments and the known structures of the most common conformations of amino acid residues, which we refer to as partial structures. To the best of our knowledge, we propose the first transformer-based model that directly utilizes experimental protein crystallographic data and partial structure information to calculate electron density maps of proteins. In particular, we use Patterson maps, which can be directly obtained from x-ray crystallography experimental data, thus bypassing the well-known crystallographic phase problem. We demonstrate that our method, CrysFormer, achieves precise predictions on two synthetic datasets of peptide fragments in crystalline forms, one with two residues per unit cell and the other with fifteen. These predictions can then be used to generate accurate atomic models using established crystallographic refinement programs.
Crystallographic and computational studies of the metal ion binding properties of parvalbumin
(2000) Cates, Mary Susan; Phillips, George N., Jr.
An astonishing number of important physiological processes are regulated by the small alkaline earth metal, calcium. Regulatory Ca2+-binding proteins must be able to distinguish Ca2+ ions in the presence of greater concentrations of other metal cations, such as Mg2+, Na+ and K+. The EF-hand family is a large class of Ca2+-binding proteins that displays this sort of preferential Ca2+-binding. The secondary and tertiary structure of the EF-hand metal ion binding site is highly conserved from one member of the family to the next. Because of this conservation, we can use the small, amenable, EF-hand protein, parvalbumin, as a model system to study the mechanisms that define the metal ion affinities and specificities of EF-hand Ca2+-binding sites in general. Our collaborator, Dr. James Potter, has designed a mutant to test directly the role of the last coordinating residue in the EF-hand binding site, the PVEF-E101D parvalbumin mutant. The crystal structures of both the Ca 2+- and Mg2+-bound complexes of PVEF-E101D have been determined. The PVEF-E101D mutant displayed a 100-fold decrease in the binding affinity for Ca2+, and the Mg2+-binding affinity was increased 10-fold. Moreover, the Ca2+ off-rate escalated from 1 s--1 in wild-type parvalbumin to 600 s--1 in the PVEF-E101D mutant. The conformation of the mutated EF-hand in the PVEF-E101D/Mg2+ structure was typical of a Mg 2+-bound EF-hand, with the exception of an F helix movement of ∼1 A toward the bound cation that allowed the shorter aspartate residue to coordinate the Mg2+ ion. The PVEF-E101D/Ca2+ structure showed that the aspartate residue is unable to bind Ca2+ in the bidentate mode normally adopted by the wild type glutamate. The resulting sixfold Ca2+ coordination in the mutant is usually characteristic of Mg2+-bound EF-hands, and this finding indicates that the binding loop is not sufficiently flexible to allow the aspartate residue to move in far enough to offer bidentate ligation of the Ca2+ ion. Two MD simulations were used to further investigate the relationship between the last coordinating residue of the EF-hand binding loop and the overall plasticity and flexibility of the loop region. The first simulation, called Alchemy, simulated the transition from Ca2+ to Mg 2+ coordination through varying the van der Waals parameters for the bound metal ions. The glutamate at position 12 was accurately and reversibly predicted to be the source of bidentate ligation of Ca2+ in our simulations. A second simulation, the Aspartate simulation, produced results that correlated well with the experimental result that an E101D substitution at EF loop position 12 resulted in monodentate Ca2+ coordination. The F helix was able to move in to the binding cavity during the simulation to allow one aspartate oxygen to bind the Ca2+ ion, but the aspartate was unable to achieve a favorable orientation for bidentate Ca 2+ coordination. The findings indicate that the interplay between the last coordinating residue of the loop, and the plasticity, or flexibility, of the binding loop, to a great extent determines the species of cations that are allowed to bind in a particular EF-hand site.
Identification and modeling of protein conformational substates
(1999) Romo, Tod Denis; Phillips, George N., Jr.
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.
Increasing the stability of recombinant adult human apohemoglobin
(2010-09-28) Olson, John S.; Phillips, George N., Jr.; Rice University; Wisconsin Alumni Research Foundation; United States Patent and Trademark Office
The disclosure relates to recombinant adult human apohemoglobin (apo-rHb) in which the stability has been increased by replacement of an amino acid with a counterpart from another organism, such as a deep sea diving mammal. This mutated apo-rHb may be more stable and/or give higher production yields than unmutated adult human apo-rHb. The mutated apo-rHb may be produced in microorganisms, such as E. coli or yeast cells, or animal erythroid cells. Some apo-rHb of the present disclosure may be used as part of a blood substitute.
Modeling protein flexibility using collective modes of motion: Applications to drug design
(2004) Teodoro, Miguel L.; Phillips, George N., Jr.
This work shows how to decrease the complexity of modeling flexibility in proteins by reducing the number of dimensions necessary to model important macromolecular motions such as the induced fit process. Induced fit occurs during the binding of a protein to other proteins, nucleic acids or small molecules (ligands) and is a critical part of protein function. It is now widely accepted that conformational changes of proteins can affect their ability to bind other molecules and that any progress in modeling protein motion and flexibility will contribute to the understanding of key biological functions. However, modeling protein flexibility has proven a very difficult task. Experimental laboratory methods such as X-ray crystallography produce rather limited information, while computational methods such as molecular dynamics are too slow for routine use with large systems. In this work we show how to use the Principal Component Analysis method, a dimensionality reduction technique, to transform the original high-dimensional representation of protein motion into a lower dimensional representation that captures the dominant modes of motions of proteins. For a medium-sized protein this corresponds to reducing a problem with a few thousand degrees of freedom to one with less than fifty. Although there is inevitably some loss in accuracy, we show that we can approximate conformations that have been observed in laboratory experiments, starting from different initial conformations and working in a drastically reduced search space. As shown in this work, the accuracy of protein approximations using this method is similar to the tolerance of current rigid protein docking programs to structural variations in receptor models.
Structural and functional analysis of proximal pocket mutants of sperm whale myoglobin
(1999) Liong, Elaine Chiu; Phillips, George N., Jr.
Myoglobin is the subject of continuing investigations because of its ability to bind oxygen reversibly. This physiological role depends on the modulation of iron reactivity and heme affinity by the globin. The hypothesis that both of these factors are affected by residues in the proximal heme pocket is tested by site-directed mutagenesis of four proximal residues: Leu89(F4), His97(FG3), His99(FG5), and Leu104(G5). The structures of several mutant myoglobins have been determined by X-ray crystallography and then used to interpret the results of functional studies. These four proximal pocket residues support and maintain the structure of the porphyrin ring via steric and/or electrostatic interactions with the heme prosthetic group. In turn, stereochemical changes in the heme group induced by a proximal pocket mutation modulate heme-iron reactivity via changes in the displacement of the iron from the heme plane: a more accessible heme iron results in greater affinity for carbon monoxide and dioxygen. Leu89 and His97 are important surface residues that protect the hydrophobic heme pocket from hydration through steric and/or electrostatic interactions with the heme. Substitutions at either position with a small or polar residue expose the heme pocket to solvent and accelerate heme loss. Ile99 and Leu104 are located in the interior region of the heme pocket beneath the heme prosthetic group. Substitution with smaller or polar residues at positions 99 and 104 also results in water penetration of the heme pocket and promotion of heme loss. Furthermore, Leu104 is located between two sites previously found to bind xenon. Crystal structures of xenon-containing Leu104 mutant myoglobins support the hypothesis that changes in the volume accessible to photodissociated ligands affects the rates of primary and secondary recombination.
Structural characterization of Methanococcus adenylate kinases
(2002) Criswell, Angela R.; Phillips, George N., Jr.
The crystal structures of adenylate kinases from the mesophile Methanococcus voltae (37°C) and the thermophile M. thermolithotrophicus (65°C) have been solved to 2.5 A resolution using molecular replacement methods. These adenylate kinases share 78% primary sequence identity, yet exhibit significantly different thermal stabilities and optimal activity ranges. Analyses of these archaeal structures reveal possible details regarding their disparate thermostabilities. In this study, we perform a comparative structural analysis of the mesophilic and thermophilic adenylate kinases and draw four general conclusions. First, we find correlation between thermostability and ionic interactions and identify a unique ionic network in the thermophilic enzyme. Second, we find beta-branched residues incorporated within alpha-helices of the thermophilic enzyme with significantly greater frequency. Third, we find examples of tighter packing within the CORE domain of the thermophilic enzyme. Last, most of the mutations in the thermophile occur very near the surface resulting in greater negative surface potential. In additional to the comparative thermostability study, we also analyze these two methanococcal structures with respect to their apparent lack of an essential lysine residue, which is present within the P-loop of previously characterized adenylate kinases. Previous modeling experiments proposed that, if protonated, His92 could participate in a similar manner as the lysine residue present in homologous adenylate kinases. From our investigation, we conclude that, as in the case of the homologous Sulfolobus acidocaldarius adenylate kinase structure, His92 is involved in interactions with the terminal phosphate group of AMP. Structural alignments of Methanococcus and homologous enzymes demonstrate that Gly14 is structurally equivalent to Lys14, indicating that an amino acid outside of the canonical P-loop must compensate for the essential lysine deficiency. We propose that this compensatory role is filled either by Arg138 or Arg140, each of which are LID domain residues conserved among the archaeal enzymes.
Structural determinants of functional behavior in distal pocket mutants of myoglobin
(1995) Quillin, Michael L.; Phillips, George N., Jr.
The physiological role of myoglobin depends on the modulation of heme activity by the protein. The hypothesis that functional properties are governed by conserved residues in the distal pocket has been tested by site-directed mutagenesis of three residues: Leu$\sp{29}$, His$\sp{64}$, and Val$\sp{68}$. To facilitate interpretation of functional data, structures of several mutant myoglobins have been determined by X-ray crystallography. Leu$\sp{29}$ controls the volume of the distal pocket. Since Val$\sp{29}$ does not contact bound ligands, this substitution does not affect ligand affinities significantly. It does permit solvent approach to the heme, thereby increasing the rate of autooxidation. Although the Phe$\sp{29}$ mutant was constructed to reduce the volume of the binding site, dipole-multipole interactions stabilize bound oxygen and reduce the rate of autooxidation substantially. His$\sp{64}$ inhibits oxygen dissociation and autooxidation by hydrogen bonding to the ligand. In conjunction with a distal water molecule, it sterically hinders carbon monoxide association. Mutation of this residue eliminates hydrogen-bonding interactions in all cases except Gln$\sp{64}$, producing low oxygen affinities and high rates of autooxidation. In the Gly$\sp{64}$ mutant, the solvent-filled distal cavity partially restores binding site polarity. In contrast, the distal pockets of Val$\sp{64}$, Thr$\sp{64}$, and Leu$\sp{64}$ are completely apolar, leading to marked increases in rates of ligand binding. Val$\sp{68}$ governs the ligand accessibility of the iron. In the Ala$\sp{68}$ mutant, only slight rate enhancements occur because the distal water molecule is retained in the deoxygenated protein. The larger side chains of Ile$\sp{68}$ and Leu$\sp{68}$ displace this water molecule and occlude the binding site in unliganded structures. The lower affinities observed in Ile$\sp{68}$ compared to Leu$\sp{68}$ are due to the decreased ability of this residue to accommodate the bound ligand. In contrast, the Phe$\sp{68}$ side chain is directed away from the iron atom and does not inhibit binding directly. Nevertheless, the reduced volume in this mutant is filled with a water molecule, retarding ligand association. In all mutants, structural perturbations are limited to the site of the substitution and the flexible corner regions of myoglobin. Furthermore, the stereochemistry of the heme-ligand complex is little influenced by changes in the distal pocket.
Structure and dynamics of Escherichia coli adenylate kinase
(1996) Berry, Michael Brandon; Phillips, George N., Jr.
Escherichia coli adenylate kinase has been studied by X-ray crystallographic and molecular dynamics approaches. The structures of E. coli adenylate kinase complexed with either AMP and AMPPNP, or AMP and ADP are presented. The AMP/AMPPNP complex has been determined to a resolution of 2.0 A, and confirms the location of the AMP and ATP binding sites on the protein. The AMP/ADP complex has been determined to resolution of 2.8 A, and demonstrates the geometry of this inhibitory complex. Molecular dynamics studies of E. coli adenylate kinase have been done using the weighted masses technique. Simulations were conducted using two general protocols (CHARMM and X-PLOR) which differ in the nature of the secondary structural constraints applied. Both protocols were successful in simulating the opening of the two mobile domains of the protein. The X-PLOR based trajectories, using a heterogeneous weighting system, were also able to simulate the closing of the mobile domains, resulting in a more Gaussian distribution of conformations than that seen in the CHARMM based method.
Structure of tropomyosin at 7A resolution
(1995) Whitby, Frank G.; Phillips, George N., Jr.
The crystal structure of tropomyosin has been determined by X-ray diffraction analysis at 7A resolution. Tropomyosin is a 400A-long muscle regulatory protein that consists of two parallel 33,000 Dalton alpha helices wound around one another to form a coiled coil and whose amino-acid sequence is characterized by a characteristic heptad repeat pattern. The structure was solved initially at 9A resolution by molecular replacement and refinement of a uniform wire model with a specially designed refinement procedure. Phase information was later derived from a single mercury derivative by single isomorphous replacement (SIR) refinement and used in the construction of an atomic model which was refined at 7A resolution. The model agrees well with the previous low-resolution X-ray structure and with models of tropomyosin in paracrystalline and micro-crystalline forms based on electron microscopy. The overall shape of the molecules, characteristics of the coiled coil and the geometry of interactions of molecules in the crystal are apparent from the structure. The molecules are precipitated by spermine and polymerize head-to-tail to form sheets of nearly parallel filaments, overlapping by about 2/3 of the molecular length in an antiparallel configuration. The relationship of two cysteine residues on each of the molecules was determined unambiguously by solving the structure of a mercury-labeled form of the protein. The shape of the molecule is influenced by local amino-acid sequence variations and crystal packing interactions. The inherent mobility of the molecule in the crystal indicates the importance of considering the flexibility and motions of tropomyosin in models of muscle thin filament regulation and cooperativity. The detailed structure of the head-to-tail overlap region cannot be ascertained from the present model, but will be an important focus of attention for future study. Through amino-acid sequence analysis, an element of quaternary structure, the coiled coil, can be directly predicted. However, tropomyosin is the largest of this class of proteins whose structure has actually been determined by X-ray crystallography.
Structures and interactions of tropomyosin with caldesmon and troponin
(1997) Hnath, Eric John; Phillips, George N., Jr.
Tropomyosin plays a central role in the regulation of skeletal, cardiac, and smooth muscle regulation. The regulatory properties of tropomyosin are mediated by its interactions with muscle-specific tropomyosin binding proteins. In skeletal and cardiac muscle the regulatory protein is troponin, while in smooth muscle the primary protein is caldesmon. The structures and interactions of tropomyosin with caldesmon, skeletal troponin, and cardiac troponin have been studied using X-ray crystallography and optical biosensors. Only whole caldesmon and the carboxyl-terminal domain of caldesmon bound tightly to tropomyosin. X-ray studies showed that whole caldesmon bound to tropomyosin in several places. Experiments with the carboxyl-terminal domain of caldesmon revealed that this region corresponded to the strongest binding site seen for whole caldesmon. Weaker association of other regions of caldesmon to tropomyosin was also observed. The structure of cocrystals of skeletal and cardiac troponin subunit T revealed that the two isoforms interacted with tropomyosin in the same general area but that cardiac troponin T bound to tropomyosin over a more extended region. The longer amino-terminal domain of the cardiac protein bound further along the carboxyl region of tropomyosin than skeletal troponin T, and the carboxyl-terminal domain of cardiac troponin T bound to tropomyosin more tightly than its skeletal counterpart. Biosensors studies of tropomyosin interacting with caldesmon and troponin measured association rate, dissociation rate, and equilibrium rate constants of these proteins for the first time. Caldesmon bound with similar affinity to several tropomyosin isoforms while troponin bound most tightly to striated muscle tropomyosin. An atomic model of tropomyosin at 5 A resolution has been constructed using a simulated annealing procedure and X-ray diffraction data from the spermine crystal form of tropomyosin. During these refinements the R-free and R were monitored. However, failure to lower the R-free suggests that this model does not accurately describe the structure of tropomyosin within these crystals. These results define interactions and structures within thin filaments of cardiac, skeletal, and smooth muscle which will be useful in elucidating the exact role of these proteins in the unique regulation of each type of muscle.
The molecular structure of green fluorescent protein
(1997) Yang, Fan; Phillips, George N., Jr.
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.
X-ray structural determination and biophysical characterization of HemAT, a chemotaxis receptor from Bacillus subtilis
(2004) Zhang, Wei; Phillips, George N., Jr.
The heme-based aerotaxis transducer (HemAT) from B. subtilis is a heme-containing protein and functions as an oxygen sensor. It can detect oxygen and transmit the signal generated from oxygen binding to regulatory proteins through its putative methyl-accepting chemotactic domain. Through other components, the signaling information is transferred to motor proteins, which control the direction of rotation of flagella and in turn lead to changes in the swimming behavior of bacteria. There is a great deal of information known about chemotaxis signaling transduction for Escherichia coli and Salmonella typhimurium. However, the detailed molecular mechanism of chemotaxis of Bacillus subtilis is in a sense reversed, because attractant binding to chemotactic receptors strengthens the activity of the downstream histidine kinase, instead of inhibiting reaction in Escherichia coli and Salmonella typhimurium. Multiple-wavelength anomalous dispersion (MAD) data were collected from crystals of HemAT using the intrinsic anomalous scatterer, iron, with synchrotron radiation. Three wavelength iron MAD data were collected to 2.8A resolution. The native data set was collected to 2.15A resolution. The crystallographic analysis reveals that the crystal belongs to P21212 1 space group with the cell dimension a = 50.00A, b = 80.12A, c = 85.95A. There are two molecules in one asymmetric unit with 40% solvent content. I have determined the crystal structures of the HemAT sensor domain in liganded and unliganded forms at resolutions of 2.15A and 2.7A. The structures show that the HemAT sensor domain is a dimeric protein with one heme group in each subunit. The structure of liganded form of HemAT sensor domain reveals a more symmetrical organization than that of the unliganded form. Tyrosine70 in one subunit shows distinct conformations in the liganded and unliganded structures. Our study suggests that disruption of HemAT symmetry plays an important role in initiating the chemotaxis signaling transduction pathway. Our kinetic and thermodynamic studies of ligand binding suggest that HemAT may employ negative cooperativity for detecting external ligand in the signal transduction. The sensor domain provides the structural evidence for such a molecular mechanism.

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