Chapter 3 of my Ph.D. dissertatation

Chapter 3

 

 

Structural consequences of axial-histidine iron bond cleavage in a selective nitric oxide sensor

 

3.1       Introduction

H-NOX domains constitute a protein family.  Members of the family are heme-based diatomic gas sensor proteins that regulate signal transduction pathways (1, 2).  Like many cell signaling proteins, H-NOX based signaling proteins are modular (3-7).  The domain modulates the activity of signaling modules such as histidine kinases in prokaryotes and guanylate cyclases in eukaryotes (3).  In Shewanella oneidensis (So) the SO2144 H-NOX domain modulates the activity of the SO2145 sensor histidine kinase (11).  In Chapter 2: “Structural studies of a selective nitric oxide sensor by solution NMR,” a series of NMR studies of the SO2144 H-NOX domain were described with the aim of addressing the following: how do H-NOX proteins bind diatomic gases at their heme iron cofactors and transduce free energy associated with ligand binding into cell signaling pathways?  Observations were described which lead to solution structures of the SO2144 protein, an NO binding H-NOX domain from So.  To address the conformational change associated with signaling, high-resolution structures in different states of activity are required.

Note:  SO2145 histidine kinase assays are reported in the dissertation of Mark S. Price (12).

In this chapter, the NMR structures of the SO2144 H-NOX protein are presented together with determinations of their effects on the cognate cell signaling partner, the SO2145 histidine kinase.  The structures are then compared and contrasted with previously derived X-ray crystal structures (8, 9, 13, 14) to merge what has been learned previously about H-NOX structural transitions and what is learned from the data presented in this thesis into a hypothesis for a structural mechanism involved in the signaling pathway.

 

3.2       Materials and Methods

3.2.1    Structure analyses

The structures in the Fe(II)-CO WT and H103G ensembles were ranked by XPLOR-NIH energy and the lowest energy structure with overall root mean squared (rms) deviations closest to the average coordinates were analyzed (Appendices 10 and 11).  Deviations of the heme from planarity were quantified by using the Normal-Coordinate Structure Decomposition (NSD) algorithm of Jentzen, Shelnutt, and coworkers (10).  The program MOLMOL was used for the calculation of rms deviations between different H-NOX structures (15).  The program DyDom was utilized for domain rotation angle calculations (16).

 

 

 

 

 

3.3      Results

3.3.1    Identification of active and inactive diamagnetic states of the SO2144 H-NOX for NMR studies

The SO2144 H-NOX and SO2145 histidine kinase form a complex (11).  The autophosphorylation activity of SO2145 is suppressed by the Fe(II)-NO form of the H-NOX SO2144 (defined as an active H-NOX state) (Figure 3.1A and 3.1B), but is not affected by the unliganded Fe(II) H-NOX (defined as an inactive H-NOX state) (Figure 3.1B). In terms of activity, the unliganded and NO-bound forms of SO2144 would be the natural states to study structurally in order to understand the basis for the activity change upon NO binding. However, the heme-iron cofactor of the SO2144 H-NOX is paramagnetic (Chapter 2).  Previous studies have shown that six coordinate Fe(II)-CO complexes are diamagnetic (17) allowing detailed NMR analysis, so efforts were focused on diamagnetic CO derivatives of SO2144. The Fe(II)-CO WT H-NOX weakly inhibits the kinase activity of SO2145, IC50 = 84 ± 5 µM, which is intermediate in activity (10-fold less-potent than the NO complex, IC50 = 9 ± 2 µM).  In proteins containing H-NOX domains, such as sGC or SO2144 H-NOX, displacement of the axial histidine from the iron has been correlated with high activity (11, 18). To mimic the state with histidine displaced from the heme, the mutation H103G was introduced, disconnecting the helix that makes contact to the iron through the sidechain. In order to isolate stable protein with heme inserted, as for other mutants of this type, it was necessary to add free imidazole to the medium.  This mutant binds CO and forms a stable diamagnetic complex (Fe(II)-CO H103G) (19, 20) (Figure 3.1C), and the inhibitory activity of this protein against the SO2145 kinase, IC50 = 22 ± 5 µM, approaches that of the WT-Fe(II)-NO complex.

 

 

The IC50 values for inhibition of histidine autophosphorylation activity for the relevant forms of protein are given in Figure 3.1D.

 

Assays of H103G were carried out in the presence of 10 mM imidazole whereas assays of the wildtype protein had no imidazole added.  Data from these two different assay conditions are comparable because control experiments done with the SO2145 histidine kinase in the absence of H-NOX show that 10 mM imidazole had no effect on kinase activity (unpublished data).  Surprisingly, NMR and kinetic data suggest that at a concentration of 10 mM, imidazole binds to the WT heme cofactor on the proximal side displacing the axial histidine from the heme iron (unpublished data). Furthermore, the 1H-15N HSQC spectra of the paramagnetic wildtype and H103G Fe(II)-NO complexes were superimposable consistent with the conclusion that the mutation and addition of imidazole did not drastically alter the structure (Figure 3.2).  Given the results described above, we focused on obtaining NMR structures of Fe(II)-CO WT (intermediately active) and Fe(II)-CO H103G (active), as they are diamagnetic and models for the Fe(II) and Fe(II)-NO forms of the SO2144 H-NOX.

 

3.3.2    NMR Structure Determination of Fe(II)-CO WT and Fe(II)-CO H103G

Fe(II)-CO WT and Fe(II)-CO H103G SO2144 H-NOX yielded 1H-15N HSQC spectra with high cross-peak dispersion, indicating that both states were stably folded (Figure 3.3).  Chemical shift assignments for both Fe(II)-CO WT and Fe(II)-CO H103G were made using standard triple-resonance coherence transfer and NOESY experiments (21) as described in detail in Chapter 2 and the results summarized here in Chapter 3.  Some assignments were additionally confirmed by amino acid specific labeling.  A majority of resonances could be assigned, the exceptions being residues 2, 20, 35, 48, 110-112, and 114.  A few crosspeaks in the 1H-15N HSQC could not be assigned due to lack of connectivities, likely arising from some of the unassigned residues.  Many of the unassigned residues are in loops and may be broadened beyond detection because of conformational exchange.  Temperature and pH dependent broadening was seen for some residues including 101, 104, 105, and 106, which are near H103, the proximal heme ligand (unpublished data).  Such broadening reflects a chemical exchange process on the intermediate to fast timescale (ms-ns).  Shifting of these peaks with pH and temperature occurred without broadening in the H103G protein, indicating faster exchange in the mutant protein.  There are titratable groups in the region affected, but no specific source of the effect could be identified.  Additionally, heme insertion isomerism about the a-g meso axis gave rise to two distinct cross-peaks for some residues within the immediate vicinity of the heme (22), with one set of peaks being considerably weaker than the other.  Weak peaks were ignored in the structural analysis.

 

The structures of both proteins were determined using NOE-derived distances, chemical shift derived dihedral angle restraints, and global orientation restraints from protein and heme derived residual dipolar couplings (RDCs).  A summary of structure statistics is provided in Table 3.1.  The 20 lowest energy refined structures from 200 random starting structures, with no NOE violations > 0.3 Å and no dihedral angle violations > 5o, were chosen to represent the solution structure ensembles of Fe(II)-CO WT and Fe(II)-CO H103G (Figure 3.4).  Average root mean squared (rms) differences from the mean for this group of 20 lowest energy structures were 0.33 Å / 0.37 Å for backbone and 0.94 Å / 0.99 Å for heavy atoms, respectively for Fe(II)-CO WT and Fe(II)-CO H103G.  The overall topology of the H-NOX domain, with seven a-helices and four b-strands as previously observed in x-ray studies of the H-NOX domains from Thermoanaerobacter tengcongensis (Tt) and Nostoc sp PCC 7120 (Ns), is preserved in the SO2144 H-NOX domain structure (8, 13, 14).

 

3.3.3    Heme environment structure refinement

For the heme axial ligands the distance between the iron atom and the axial histidine nitrogen and CO carbon were fixed at 2.2 Å and 1.9 Å, respectively, based on EXAFS data from Fe(II)-CO myoglobin and X-ray crystallographic data for the Fe(II)-CO complex of the Ns H-NOX domain (14, 23).  The NMR data do not give direct information about the orientation of the heme bound CO ligand.  In the calculated structures the Fe-C-O bonding was modeled as linear based upon structures of small molecule heme models, X-ray crystallographic data derived from the Fe(II)-CO H-NOX from Ns, and X-ray crystallographic and spectroscopic data from Fe(II)-CO myoglobin (14, 24-27).  Because no unambiguous NOE distance restraints could be identified between the propionates of the heme cofactor and the protein, loose (i.e. 5Å) distance restraints were utilized to restrain the position of the heme propionate groups so as to preserve their expected electrostatic contacts with the strictly conserved YxSxR motif.  The use of these restraints is justified by the observations that (i) the YxSxR motif is strictly conserved in the H-NOX family, (ii) crystallographic data display heme-YxSxR hydrogen bonds in two different prokaryotic H-NOX domains, and (iii) they do not cause violation of any of the experimental NMR geometric restraints (8, 14).

 

Table 3.1:  Structure statistics                                                                                                                         

 

A.  Experimental restraints                                                               Fe(II)-CO WT                   Fe(II)-CO H103G    

Distance restraints

Total NOE distance restraints                           3112                                       2222

Long range  | i – j | > 4                                         717                                         562

Medium range 1< | i – j | < 5                               678                                         498

Sequential  | i – j | = 1                                           706                                         565

Intraresidue                                                          941                                         547

Heme-protein                                                       70                                           48

Imidazole-protein                                                                N/A                                         2

Hydrogen bonds                                                  150                                         148

Dihedrals

TALOS (φ/ψ)                                                        277                                         250

 

Dipolar coupling restraints

HN                                                                          129                                         114

HACA                                                                    133                                         114

HC heme                                                               4                                              3

 

B.  Measures of structure quality                                                     Fe(II)-CO WT                     Fe(II)-CO H103G 

Deviations from experimental restraints

Violations per structure / RMS Deviation                                                                                                                         NOE ( > 0.3 Å )                                                                   0 / 0.023                                                0 / 0.026

Dihedrals ( > 5º )                                                                0 / 0.587                                                0 / 0.357

Residual dipolar couplings R factor / RMS Deviationa

HN Rdip /(Hz)                                                                      10.9 % / 1.32                        9.6 % / 1.04

HACA Rdip /(Hz)                                                                15.2 % / 1.39                        15.8 % / 1.89

Heme Rdip /(Hz)                                                                 0.6 % / 0.15                         1.7 % / 0.26

Deviations from idealized covalent geometry (protein only)                                                                                                      Bond (Å)                                                                               0.005                                      0.004

Angle (º)                                                                 0.471                                      0.596

Improper (º)                                                                          0.327                                      0.378

 

Average coordinate RMSDs to the mean (residues 5-30, 46-109, 120-178)

Backbone (Å)                                                                       0.33                                        0.37                                        Heavy Atoms (Å)                                                                                0.94                                        0.99

 

Structure quality factorsb, Z-scoresc

Procheck G-factor (phi / psi only)                                    -1.34                                       -1.62

Procheck G-factor (all dihedral angles)                           -2.54                                       -2.52

Verify3D                                                                               -1.15                                       -1.37

ProsaII (-ve)                                                                         0.02                                       -0.25

MolProbity clashscore                                                        -5.40                                       -5.46

 

Ramachandran plot summary (%)

Most favored regions                                                          83.3                                        82.9

Additionally allowed regions                                             13.7                                        14.2

Generously allowed regions                                               2.5                                          2.4

Disallowed regions                                                               0.5                                          0.5

 

 

a  Rdip isdefined as the ratio of the root mean squared (rms) difference between observed and calculated RDC values to the rms difference if the bond vectors were distributed randomly (28) and was calculated using the iDC toolkit (29).

 

b  Determined using Protein Structure Validation Suite version 1.3 (30).  The suite includes Procheck (31), Verify3D (31), ProsaII (32) and MolProbity (33).

 

c   With respect to mean and standard deviation for a set of 252 X-ray structures < 500 residues, of resolution <= 1.80 Å, R-factor <= 0.25 and R-free <= 0.28; a positive value indicates a ‘better’ score.

 

 

 

 

 

A two step simulated annealing protocol was utilized to determine conformations of the SO2144 H-NOX heme cofactor consistent with all the NMR data.  The protocol consisted of (i) a high temperature step to determine the overall protein fold by annealing the protein from an extended random chain around a heme cofactor rigidly locked in a planar configuration and (ii) a low temperature annealing step with only a weak planarity restoring potential on the heme cofactor so that it adopts a planar configuration in the absence of protein derived van der Waals contacts or NMR derived geometric restraints, but can sample non-planar conformations as required to satisfy the protein and heme NMR constraints (34). Although the heme planarity restoring potential may not accurately reflect the quantum mechanical potential energy surface of heme deformations, the focus of the protocol is to allow steric contacts between the heme and side chains that abut the heme to distort it from planarity.  While the heme distortions seen in the NMR structure ensembles are not highly determined by the NMR data, the differences between the Fe(II)-CO WT and Fe(II)-CO H103G ensembles are significant and reflect changes in the van der Waals contacts between the heme pocket and the porphyrin.  A similar strategy has been successfully employed to characterize the heme cofactor and heme pocket van der Waals contacts in Fe(II)-CO myoglobin (35).

 

3.3.4    Conformational differences between Fe(II)-CO WT and Fe(II)-CO H103G

To obtain an initial assessment of the regions with conformational differences between the Fe(II)-CO WT and Fe(II)-CO H103G, the differences in backbone amide and side chain methyl chemical shifts were quantified (Figure 2.29A and 2.29B) and mapped onto the structure of Fe(II)-CO WT.  Affected residues are found primarily in regions of secondary structure that immediately flank the proximal face of the heme cofactor.  The largest changes are located in residues 100-115 that comprise helix aF and the turn that connects helix aF to strand b1.  Smaller changes were seen in sites more peripheral to the heme, such as residues D86 and K87 in aE and I118 in b1.  The heme cofactor makes significant contributions to chemical shifts of residues within a ~10 Å distance from the heme iron (36).  The magnitudes and locations of chemical shift changes between Fe(II)-CO WT and Fe(II)-CO H103G are expected to primarily reflect a change in the position of the proximal helix aF relative to the heme cofactor of the H-NOX domain.  The absence of chemical shift changes in the distal sub-domain suggests that the heme cofactor position does not change relative to this half of the molecule.

 

To understand the nature of changes occurring upon activation of the H-NOX, a difference distance matrix was examined.  Matrix plots based on differences in a-carbon distances were calculated with the program DDMP (Center for Structural Biology at Yale University and Figure 3.5) for the single structure closest to the average structure in each ensemble (Fe(II)-CO WT and Fe(II)-CO H103G).  The distances which changed significantly were between the subdomains on opposite sides of the heme. When the proximal subdomains of structures in the Fe(II)-CO WT ensemble were superimposed (using residues 100-110 and 120-178 as shown in Figure 3.6) the average pairwise RMSD of the backbone of the superimposed residues was 0.48 Å, and for the Fe(II)-CO H103G ensemble it was 0.58 Å.  When these same residues are superimposed between the two ensembles the average pairwise RMSD is 0.72 Å (Figure 3.6) showing that there is little change within this subdomain.  Within these ensemble superpositions the average pairwise RMSD of the distal subdomain, residues 5-30 and 46-99, is 0.88 Å for Fe(II)-CO WT and 0.74 Å for Fe(II)-CO H103G.  The average pairwise RMSD of the distal subdomain between ensembles is 1.35 Å.  These observations indicate that the conformational change is primarily a rigid body displacement of the distal half of the molecule relative to the proximal half, with translation increasing in magnitude with the distance from the domain interface.

 

 

3.3.5    Heme environment

The position of the heme cofactor was well-defined in both the Fe(II)-CO WT and Fe(II)-CO H103G structures through 70 heme-protein NOE distance restraints in Fe(II)-CO WT and 48 heme-protein and 2 imidazole-protein NOE restraints in Fe(II)-CO H103G.  In particular, 16 (Fe(II)-CO WT) and 15 (Fe(II)-CO H103G) NOE distance restraints from sites in the protein to the methine protons of the heme ring greatly restrict the position of the heme cofactor during the structure calculation process.  13C-1H residual dipolar couplings measured using a protein sample with the heme specifically labeled at the methine positions with 13C define the orientations in the heme protein molecular frame of the C–H bond vectors that are part of the heme ring (37, 38).  Nonplanar heme conformations better represent the solution configuration of the heme cofactor since structures calculated with a planar heme cofactor fit the methine CH RDCs (Fe(II)-CO WT / Fe(II)-CO H103G) with a Rdip, heme methine of 13.2% / 17.1% while structures calculated allowing the heme to sample nonplanar configurations reduces Rdip,heme methine to 0.6% / 1.7% (28).  In contrast, protein backbone Rdip (derived from HN and HαCα RDCs) did not change to a significant extent with the enforcement of heme planarity.

 

Figure 3.7 shows the heme region from the Fe(II)-CO WT and Fe(II)-CO H103G structures.  The distal heme pocket is lined with aliphatic and aromatic residues as shown in Figure 3.7A-C (I5, L77, L145).  Deviations of the heme from planarity were characterized by using the Normal-Coordinate Structure Decomposition (NSD) algorithm

 

 

 

 

 

of Jentzen, Shelnutt, and coworkers (10).  The average amplitudes for specific distortion modes are shown for each ensemble in Figure 3.7D.  Particularly striking differences between Fe(II)-CO WT and Fe(II)-CO H103G are observed in the ruffling and doming modes.  On average there is less heme doming and ruffling in Fe(II)-CO H103G than in Fe(II)-CO WT, that is the heme becomes more planar in the kinase-inhibitory conformation.  The plots shown in Figure 3.7E and Figure 3.8 illustrate changes in the median and width of the distribution of heme distortions for the NMR structure ensembles.  The most significant difference is seen in the doming distribution; more structures in the Fe(II)-CO WT ensemble show doming greater than 0.5 Å than in Fe(II)-CO H103G (see Figure 3.8 for the complete results of this analysis).

 

3.3.6    Structural consequences of histidine displacement from the heme iron

Superimposing the NMR derived structures of Fe(II)-CO WT and Fe(II)-CO H103G in the regions containing residues 100-110 (aF) and 120-178 (aG and b1-4) reveals a rotation of the distal sub-domain of the protein relative to the proximal, by about 4°(Figure 3.9A).  Changes in relative positions of residues around the heme in the H-NOX domain are associated with this rotation.  For the following discussion the structural changes are quantified by taking the average distance change between specific atomic positions in the NMR ensembles, reported with the standard deviation for the values in the ensemble.  Four distal heme pocket residues, including I5, V8, L73, and L145 in helices aA-D and aG, are constrained by a dense network of NOE distance restraints in both Fe(II)-CO WT and Fe(II)-CO H103G.  Distal heme pocket conformational changes upon activation are relatively small.  For example, as the distal sub-domain pivots about aD the distance between the g carbon of L73 and the b carbon of V8 changes from 4.5 ± 0.04 Å in Fe(II)-CO WT to 5.5 ± 0.04 Å in Fe(II)-CO H103G (Figure 3.9B).  The observed pivoting motion is required to accommodate flattening of the heme cofactor.

 

Three distance changes in the heme pocket capture the essence of the conformational change following displacement of the axial histidine from the iron (Figure 3.9C).  First, the proximal helix aF moves away from the heme following rupture of the iron-histidine bond.  The distance between the a carbon of residue 103 and the heme iron is 6.2 ± 0.03 Å in Fe(II)-CO WT and 8.1 ± 0.3 Å in Fe(II)-CO H103G.  Second, in the Fe(II)-CO WT structure the heme is held against P116 by the Fe-H103 coordination resulting in a strongly domed heme configuration.  The removal of the His sidechain produces a change in Fe(II)-CO H103G, releasing proximal helix aF from coordination to the heme iron.  Removing the His-Fe interaction results in heme flattening as the van der Waals clash between P116 and the heme A ring is relieved.  This change can be quantified by the distance between the g carbon of P116 and the carbon at the vertex of propionate A and heme pyrrole ring A.  This distance is 3.9 ± 0.3 Å in Fe(II)-CO WT and is 5.4 ± 0.7 Å in Fe(II)-CO H103G.  Third, the distance between the terminal methyl carbon of I5 and methine carbon D of the heme is similar in Fe(II)-CO WT and Fe(II)-CO H103G, 6.1 ± 0.3 and 6.3 ± 0.4 Å, respectively.  Thus, the heme and the distal sub-domain move in concert away from proximal helix aF.

 

 

3.4       Discussion

In this study, we characterized a change that occurs in the SO2144 H-NOX domain that may lead to inhibition of the SO2145 histidine kinase.  Two solution structures were solved.  The first structure was that of Fe(II)-CO WT.  Two lines of evidence suggest that the structure of Fe(II)-CO WT is in a conformation resembling, but not identical to, the “inactive” Fe(II) state.  First, crystallographic data from the Ns H-NOX domain provided high resolution structures of a non-oxygen binding H-NOX domain in the Fe(II), Fe(II)-CO, and Fe(II)-NO (6 coordinate) states (14).  Superpositions of the Ns Fe(II) and Fe(II)-CO crystal structures revealed a subtle < 1 Å pivoting motion of the heme cofactor about an axis perpendicular to the plane of the pyrrole D ring.  However, no large-scale conformational changes in the relative positions of the distal and proximal subdomains were observed.  As a result, the Fe(II) and Fe(II)-CO Ns structures may represent the structural consequences associated with the binding of CO to the Fe(II) unligated SO2144 H-NOX domain.  Second, reminiscent of sGC in eukaryotes, binding of CO to the SO2144 H-NOX domain modulates the kinase activity of SO2145 but to lesser extent than NO (11, 39).  One possible explanation for the observation of weak activation upon CO binding in both enzymes is that, reminiscent of the Perutz model for hemoglobin allostery, the change in iron spin state upon the transformation of Fe(II) WT to Fe(II)-CO WT following CO association may result in the movement of the heme iron from a puckered position below the heme plane into the plane of the heme (40).  Consistent with this model, comparison of the Fe(II) and Fe(II)-CO the crystal structures of the Ns H-NOX domain shows that the iron moves into the plane of the porphyrin following the binding of CO (14).

 

The second structure investigated in this study was that of Fe(II)-CO H103G.  The portions of the NMR spectra of Fe(II)-NO H103G and Fe(II)-NO WT, where resonances are not broadened beyond detection, are identical.  This observation suggests that the H103G mutant is quite similar to the WT domain and does not lead to the domain adopting a nonphysiological conformation.  As with sGC, recombinant expression of H103G in the absence of imidazole yields apoprotein consistent with H103 as the heme axial ligand (20).  Biochemical evidence presented in this work supports the hypothesis that this structure represents an “active” conformation of the H-NOX domain, resembling Fe(II)-NO.  Similar to Fe(II)-NO WT, the Fe(II) and Fe(II)-CO states of the H103G mutant inhibit the SO2145 histidine kinase, indicating the H103G mutant shifts the domain into a conformation resembling that of Fe(II)-NO WT.

 

Two distinct conformations of the H-NOX domain from Tt in the Fe(II)-O2 WT state were observed in X-ray crystal structures derived from monoclinic crystals (8).  These structures reveal that the degree of heme planarity is coupled to a rotation of the proximal half of the H-NOX domain relative to the distal half.  A conformational change of a similar nature is observed by comparison of the NMR structure ensembles of Fe(II)-CO WT to Fe(II)-CO H103G.  The residue network responsible for transmitting changes in heme planarity into large-scale conformational changes in the rest of the protein is conserved across H-NOX domains (Figure 3.10) (8).  Furthermore, between the Tt and So H-NOX domains, key residues that induce heme distortion are strictly conserved.  For example, P115, L144, and I5 in the Fe(II)-O2 WT Tt protein maintain the distortion of the heme through van der Waals interactions at positions flanking the heme proximal and distal faces, respectively.  Within the tertiary structure of the SO2144 H-NOX domain, residues P116, L145, and I5 are positioned equivalently (Figure 3.7C).  In the Fe(II)-CO WT and Fe(II)-CO H103G structures presented here the heme cofactor flattens following the cleavage of the axial-iron bond, providing evidence that changes in heme planarity are an important component of the signaling mechanism of these domains.  Concomitantly, the heme cofactor and N-terminal subdomain move as a single body away from helix αF.

 

Myoglobin samples in solution and in crystals have very similar spectroscopic features and binding kinetics parameters suggesting that the protein is conformationally similar in these states (35).  Similarly, X-ray crystallographic and resonance Raman studies have provided evidence that the heme cofactors in H-NOX proteins adopt nonplanar conformations in both crystalline and solution phases (8, 9, 41).  The X-ray crystal structures of both wild type and mutant H-NOX domains have been determined in non-isomorphous crystal forms (8, 9, 13, 14).  These structures provide a dataset within which to compare the structures of closely related H-NOX domains in different crystal packing/lattice environments.  Across this dataset, a range of different protein and heme conformations are observed, thereby suggesting the nature of the conformational mobility responsible for signal transduction controlling enzyme activity.  As quantified in Table 3.2, NSD of the heme cofactors from these structures shows that they adopt a range of conformations in crystals, with each conformation exhibiting different normal mode contributions.  For example, the major contributors to heme distortion in the H-NOX domain from Ns are ruffling and doming (14).  In contrast, the major contributors to heme distortion in wildtype Tt H-NOX are saddling and ruffling (9).  Pellicena et al. carried out molecular energy minimizations and concluded that the P115 residue (P116 in So), strictly conserved across the H-NOX protein family, contributes to heme distortion (8).  When this residue is mutated to alanine in the Tt H-NOX domain, thereby reducing the steric bulk at this position, the heme adopted a flatter conformation as quantified by rms deviation from planarity (9).  Within each asymmetric unit cell of the P115A crystal lattice four distinct Fe(II)-O2 complexes (A-D) were observed.  Since the range of conformations seen in the P115A Tt H-NOX domains structures are representative of the range of conformations seen in X-ray crystal structures of H-NOX domains, we focus discussions and comparisons of H-NOX crystal structures (9) to NMR structures on this set of structures.  Each of the P115A Tt molecules exhibited different amounts of heme distortion (Table 3.2) that are directly correlated with changes in the overall conformation of the protein (9).  When the C-termini of the four P115A structures are overlaid together with a representative wild type Tt H-NOX structure (Fe(II)-O2 monoclinic, molecule A) (Figure 3.11A) different conformations of the N-terminal subdomain, reminiscent of a bending hinge, become apparent.  This hinge bend motion is also seen in other H-NOX crystal structures (Table 3.3).  A hinge bending motion about αD similar to that seen in the C-terminal overlay of the Fe(II)-CO WT and Fe(II)-CO H103G So H-NOX solution structures (Figure 3.11B) is observed.

 

 Table 3.2:  NSD analysis of hemes in H-NOX domain X-ray crystal structures.a

 

Speciesb

Protein, Crystal form

Heme

PDB Code

Molecule

Saddling (B2U)

(Å)

Ruffling (B1U)

(Å)

Doming (A2U)

(Å)

Tt

WT, monoclinic

Fe(II)-O2

1u55 (8)

A

-1.08

-1.11

-0.10

B

-0.65

-0.79

0.32

Tt

WT

Fe(III)

1u56 (8)

A

-1.11

-1.17

-0.15

B

-0.71

-0.55

0.21

Tt

WT, orthorhombic

Fe(II)-O2

1u4h (8)

A

-1.09

-1.09

-0.02

B

-1.00

-1.20

0.10

Ns

WT

Fe(II)

2o09 (14)

A

-0.14

-0.42

0.48

B

-0.01

-0.31

0.53

Ns

WT

Fe(II)-NO

2o0c (14)

A

-0.15

-0.69

0.49

B

-0.04

-0.42

0.55

Ns

WT

Fe(II)-CO

2o0g (14)

A

-0.35

-0.58

0.43

B

-0.21

-0.45

0.43

Tt

P115A

Fe(II)-O2

3eee (9)

A

-0.40

-0.77

0.04

B

-0.50

-0.61

0.03

C

-0.04

-0.49

-0.09

D

0.07

-0.52

-0.03

Tt

WT

Fe(II)-O2

1xbn (13)

A

-0.71

0.66

-0.19

 

aHeme conformations calculated using NSD (10).

bTt” – Thermoanaerobacter tengcongensis, “Ns” – Nostoc sp PCC7120.

Table 3.3:  Protein backbone atom deviation of Tt H-NOX X-ray crystal structures.a

 

Protein

Heme

PDB Code

Molecule

 

N- and C-terminal

subdomain

rotation angleb,e

(o)

N-terminal rms deviation

(Å)c,e

Overall rms deviation (Å)d,e

P115A

Fe(II)-O2

3eee (9)

A

4.4

1.65

1.13

P115A

Fe(II)-O2

B

4.0

1.45

0.98

P115A

Fe(II)-O2

C

6.9

2.02

1.39

P115A

Fe(II)-O2

D

8.8

2.94

1.98

WTf

Fe(II)-O2

1u55 (8)

A

WT

Fe(II)-O2

B

9.0

2.46

1.65

WTg

Fe(II)-O2

1u4h

A

5.6

1.58

1.13

WT

Fe(II)-O2

B

2.4

0.65

0.46

WT

Fe(III)

1u56

A

0.8

0.28

0.26

WT

Fe(III)

B

8.0

2.25

1.52

WT

Fe(II)-O2

1xbn (13)

A

13.5

3.89

2.62

 

aThe Fe(II)-O2 monoclinic Molecule A wild-type (PDB code: 1U55) Tt H-NOX was used for comparisons in Table 3.3.

bThe N-terminal domain was defined as residues 5-30 and 47-83 and the C-terminal domain was defined as residues 100-105 and 119-178.

cTt H-NOX residues 5-30 and 47-83 were used for N-terminal rms deviation.

dResidues 5-30, 47-105, and 119-178 were used for overall rms deviation.

ePrior to calculations residues 100-105 and 119-178 were aligned.

fMonoclinic.

gOrthorhombic.

 

The hinge bending motion observed in Tt H-NOX domains is correlated with heme cofactor planarity.  A comparison of the heme cofactors of Tt P115A molecule D and wild type Tt H-NOX molecule A from a monoclinic crystal form is shown in figure 3.12 (9).  The results of the P115A heme cavity mutation are profound.  Decreasing the size of a bulky protein residue in close van der Waal contact of the heme signifigantly reduces the heme distorting force exerted by the protein scaffold.  In the wild type protein, the invariant P115 compresses the heme at the pyrrole D ring, inducing a pivoting of the attached proprionate group.  Upon alanine substitution, the heme pyrrole D ring moves back into the heme plane reducing the degree of pivoting of the associated proprionate.  In the So H-NOX domain, the heme is inserted into the protein in the opposite orientation about the a-g meso axis relative to the Tt H-NOX domain.  Thus, pyrrole ring A is positioned analogously in the So H-NOX protein.  Referring back to Tt H-NOX comparison, heme flattening triggers a rearrangement in the relative positions of distal and proximal heme pocket residues.  In particular, I5 in close contact with pyrrole ring A shifts its position relative to the positions of the other residues in the heme pocket.  The corresponding shift in the position of I5 induces a displacement of αA.  Shifting of αA propagates a large-scale rotation of the N-terminal subdomain relative to the C-terminal subdomain (Figure 3.11A and 3.12); the N-terminal sub-domain pivots about αD leading to a 3.8 Å rms deviation (residues 1-83) with respect to the fixed C-terminus (Figure 3.12) (9).  Thus, the heme wedged between the N and C terminus acts as a pivot point whose small deviations away from planarity are amplified into large scale N and C terminal subdomain rotations.

 

With respect to the So H-NOX domain (Figure 3.9) NMR structures, a similar hinge bending motion is seen following axial-iron bond cleavage with the subtle difference that the heme translates away from αF by approximately 2 Å.  Here, we perform a similar analysis of the large scale rotation of the N-terminal subdomain relative to the C-terminal subdomain seen in Fe(II)-O2 Tt WT and P115A crystal structures (8, 9) (Table 3.3) and in Fe(II)-CO WT and H103G So NMR structures (Table 3.4).  The conformational change is quantified by N-terminal rms deviation and rotation angle when their C-terminal subdomains are aligned.  From this analysis, it becomes clear that the conformational differences between Tt Fe(II)-O2 WT monoclinic molecule A (PDB code: 1u55) (8) and Tt Fe(II)-O2 P115A molecule A (PDB code: 3eee) (9) (Figure 3.13) is most analogous to the conformational differences observed between the Fe(II)-CO WT and Fe(II)-CO H103G So H-NOX domain with respect to the relative orientations of the N and C termini.  Since the heme cofactor in the NMR structures presented here displays a wide range of conformations, all consistent with the NMR data, we do not tabulate the heme distortion as done with the H-NOX X-ray crystal structures in Table 3.2.  Rather we choose to discuss the distribution of the heme structures as presented in box plot format in figure 3.8 or as averages when the context is appropriate.

 

Table 3.4:  Protein backbone atom deviations of Fe(II)-CO WT from Fe(II)-CO H103G So H-NOX NMR solution structures.a

 

Protein

Heme

N- and C-terminal

subdomain

rotation angleb,e

(o)

N-terminal rms deviation

(Å)c,e

 

Overall rms deviationd,e (Å)

WT

Fe(II)-CO

4.0

1.87

1.37

 

aThe structure closest to the average backbone structure of each NMR structure bundle were used for this calculation.

bThe N-terminal domain was defined as residues 5-30 and 46-82 and the C-terminal domain was defined as residues 100-106 and 120-178.

cResidues 5-30 and 46-82 were used for N-terminal rms deviation.

dResidues 5-30, 46-106, and 120-178 were used for overall rms deviation.

ePrior to calculations residues 100-106 and 120-178 were aligned.

 

 

 

 

H-NOX domain heme distortions are caused primarily by van der Waals interactions in the heme cavity with a strictly conserved proline (P116 in So and P115 in Tt) seeming to play a particularly important role.  Multiple sequence alignments indicate that this residue along with the axial histidine ligand (H103 in So and H102 in Tt) is strictly conserved across the entire family, suggesting that these two residues may work together to carry out conformational changes associated with signaling.  In all H-NOX structures solved to date these two residues are packed together against the proximal heme face (Figure 3.14) (8, 9, 13, 14).  In the context of NO signaling, the histidine changes its coordination to the heme iron (Chapter 1).  In the absence of NO the histidine is bound to the iron; binding of NO triggers its release.  In this work, to test the structural consequences of the Fe-bond breakage we mutated H103 to a glycine residue to study the effects of Fe-bond cleavage on H-NOX structure.  This mutation leads to changes in activity of the H-NOX domain that resemble those that occur when NO binds to the domain.  Structural observations presented in Figures 3.5-3.9 suggest that this mutation leads to an overall flattening in the heme cofactor that initiates a hinge movement of the N-terminal subdomain with respect to the C-terminal subdomain.  In these structures, the heme pivots about P116 as it flattens.  Thus, P116 and H103 may work together with the P116 residue acting as a molecular wedge, priming the heme into a strained conformation poised for conformational change.  In the proposed model, a conformational change is initiated when H103 dissociates from the heme iron following the binding of NO.  Like a compressed spring, the heme stores energy required to change conformation.  When the H103G mutation is introduced, the protein no longer compresses the heme as tightly and the signal transduction capability is compromised as the domain is not longer able to interconvert between active and inactive conformations.

 

Previous NMR studies in several well studied protein systems provide a context for the interpretation of the current NMR solution structures of the So H-NOX domain.  In T4 lysozyme, a protein that undergoes an analogous hinge bend conformational change as seen in the H-NOX domain, RDCs have been utilized to determine which X-ray structure of this protein best resembles the solution structure.  The studies of T4 lysozyme provide a benchmark for comparing protein structures derived from these two techniques (Appendix 12).  Unfortunately, this cannot yet be done with H-NOX domains because structural data provided by NMR and X-ray crystallography have not yet been collected on an H-NOX domain whose signal transduction activity has also been simultaneously measured.

An alternative application of RDCs is to use them together with other (independent) NMR observables such as NOEs, coupling constants, and chemical shifts to calculate structures de novo.  In this thesis, I utilize this approach.  The NMR data were used to derive the solution structures of the Fe(II)-CO WT and Fe(II)-CO H103G SO2144 H-NOX domain from So.  The data presented here support the model that heme distortion is involved in the conformational change.  First, the positions of the methine carbon-hydrogen bonds make residual dipolar couplings, indicators of bond orientation in the alignment frame (42, 43), able to distinguish between planar and non-planar configurations of the meso positions of the heme.  Non-planar heme configurations better fit the residual dipolar couplings than planar configurations.  Second, residues that are within van der Waals contact of the heme cofactor are restrained by NMR data derived from RDCs, NOE distance restraints, and chemical shift dihedral angle restraints.

 

In analogy to the structure determination of Murphy et al. of the WT and M9I mutant human SRY domain-DNA complex (44), the Fe(II)-CO WT and Fe(II)-CO H103G structure ensembles were refined with similar NMR derived geometric restraints.  An equivalent number of restraints of similar types (e.g. NOEs, RDCs, etc.) were utilized to calculate the Fe(II)-CO WT and H103G So H-NOX domain solution structures and the conformation of the heme.  In the SRY domain-DNA domain structure determination, the large number of protein and DNA restraints, in particular RDCs, used to calculate structures were sufficient to resolve subtle differences in the conformation of the bound DNA.  Analogously, in the Fe(II)-CO WT and Fe(II)-CO H103G solution structures presented in this chapter, the large number of NMR restraints were able to resolve differences in heme and protein conformation.  For example, the global orientations of the N and C terminal subdomains of the H-NOX protein are defined by RDC data derived from atoms in the protein backbone (HN and HαCα RDCs).  Since in the structure calculation process the heme is not constrained to be flat, the protein NMR restraints are able to define the conformation of the bound heme cofactor if they are within van der Waals contact of the heme ring.  Finally, similar to a residual dipolar coupling based structure ensemble of ubiquitin (45), the width of the ranges of heme distortions observed in the Fe(II)-CO WT and Fe(II)-CO H103G structure ensembles are similar to the width of the ranges of heme distortions observed in X-ray crystal structures of other H-NOX domains (Figure 3.8) (8, 9, 13, 14).  Thus, the differences observed between the two So NMR structure ensembles (Fe(II)-CO WT versus Fe(II)-CO H103G) suggest they are a reflection of conformational differences in the H-NOX protein scaffold induced by changes of the heme cofactor.  Furthermore, the NMR structures suggest that the heme may sample different conformations similar to those observed in crystal structures of other H-NOX domains in solution.  This observation supports the conclusion that heme conformational changes inferred from X-ray structures are functionally relevant.

 

The finding that the removal of the axial histidine iron bond from the H-NOX domain initiates a rotation of the distal sub-domain relative to the proximal suggests a model for the mechanism of conformational transition between the unligated Fe(II) and Fe(II)-NO states.  The model, which will hereafter be called the “heme strain model” proposes that, in the unligated Fe(II) state, the heme iron is coordinated by the H103 axial heme ligand (Figure 3.15).  In this state the heme is held tightly against P116, resulting in a steric clash between the heme macrocycle and the proline ring.  In order to relieve strain, the heme distorts away from planarity.  When NO binds to the heme, H103 is released from coordination to the iron due to the “trans effect” (46).  With the axial histidine-iron bond broken, the heme can relax into a conformation that relieves the strain associated with close apposition to P116.  The heme moves away from proximal helix aF and concomitantly flattens.  Finally, the distal sub-domain follows the trajectory of the heme, rotating away from proximal helix aF about a pivot point formed by the residue G70 of helix aD and G144 of helix aG.  Strong support for the involvement of heme distortion in the conformational transition between the inactive unligated Fe(II) and active Fe(II)-NO conformations derives from previously determined X-ray crystallographic structure of H-NOX domains (8, 9, 13, 14).  In those studies, structural comparisons between different crystal forms of one particular H-NOX ligation state highlighted a region of conformational plasticity similar to that observed in the Fe(II)-CO WT and Fe(II)-CO H103G solution structures, and thus provided a potential mechanism for how heme flattening leads to a transition in the relative orientation of the distal and proximal halves of the H-NOX domain.  Here we extend those observations by providing both structural and biochemical evidence that correlates release of the axial histidine heme ligand with changes in heme planarity and H-NOX domain structure in solution.  How these conformational changes modulate the activity of associated signaling domains awaits further investigation.

 

Figure 3.15:  Model of the conformational changes that occurs in the H-NOX domain upon the binding of NO to the unligated Fe(II) state.

3.5       Summary

In summary, the observations presented here are consistent with the emerging role for heme distortion in protein function.  Two solution structures, Fe(II)-CO WT and Fe(II)-CO H103G, of the So H-NOX domains are derived in different states of activity.  The conformational changes observed between these two states resemble those previously observed fortuitously in crystals (H-NOX domains from Tt and Ns) (8, 9).  Ultimately, systematic studies of the So H-NOX domain by X-ray crystallography and NMR under similar conditions (e.g. oxidation state, ligands, pH, temperature, etc.) may provide higher resolution insights into the mechanism by which the heme cofactor of the H-NOX domain acts as a pivot facilitating a hinge bending motion between the N and C terminal subdomains of the protein.  However, the data presented here suggest that the conformation of the heme is a component of the mechanism by which these signaling modules regulate the activity of downstream signaling enzymes.  The structures presented here, when interpreted together with previously derived X-ray structures of the H-NOX domain, suggest a new mechanism by which the heme cofactor can be used to carry out a cellular function.

 

3.6       Acknowledgments

            I thank Joey Davis for help with protein preparations; Douglas Mitchell for help with mass spectrometry; Jeffery Pelton for help with NMR spectroscopy, Mark Price for performing histindine kinase assays, Milton Werner (Rockefeller University) for help with residual dipolar coupling measurements; and James Chou (Harvard University) for help with residual dipolar coupling measurements and structure calculations.

 

3.7       References

1.         Boon EM & Marletta MA (2005) Ligand specificity of H-NOX domains: from sGC to bacterial NO sensors. J Inorg Biochem 99:892-902.

2.         Boon EM & Marletta MA (2005) Ligand discrimination in soluble guanylate cyclase and the H-NOX family of heme sensor proteins. Curr Opin Chem Biol 9:441-446.

3.         Iyer LM, Anantharaman V, & Aravind L (2003) Ancient conserved domains shared by animal soluble guanylyl cyclases and bacterial signaling proteins. BMC Genomics 4:5.

4.         Galperin MY (2004) Bacterial signal transduction network in a genomic perspective. Environ Microbiol 6:552-567.

5.         Stock AM, Robinson VL, & Goudreau PN (2000) Two-component signal transduction. Annu Rev Biochem 69:183-215.

6.         Pawson T & Nash P (2003) Assembly of cell regulatory systems through protein interaction domains. Science 300:445-452.

7.         Lim WA (2002) The modular logic of signaling proteins: building allosteric switches from simple binding domains. Curr Opin Struct Biol 12:61-68.

8.         Pellicena P, Karow DS, Boon EM, Marletta MA, & Kuriyan J (2004) Crystal structure of an oxygen-binding heme domain related to soluble guanylate cyclases. Proc Natl Acad Sci U S A 101:12854-12859.

9.         Olea C, Boon EM, Pellicena P, Kuriyan J, & Marletta MA (2008) Probing the function of heme distortion in the H-NOX family. ACS Chem Biol 3:703-710.

10.       Jentzen W, Ma JG, & Shelnutt JA (1998) Conservation of the conformation of the porphyrin macrocycle in hemoproteins. Biophysical Journal 74:753-763.

11.       Price MS, Chao LY, & Marletta MA (2007) Shewanella oneidensis MR-1 H-NOX regulation of a histidine kinase by nitric oxide. Biochemistry 46:13677-13683.

12.       Price MS (2008) Investigations into a novel two component signaling pathway in Shewanella oneidensis MR-1. (University of California, Berkeley).

13.       Nioche P, et al. (2004) Femtomolar sensitivity of a NO sensor from Clostridium botulinum. Science 306:1550-1553.

14.       Ma X, Sayed N, Beuve A, & van den Akker F (2007) NO and CO differentially activate soluble guanylyl cyclase via a heme pivot-bend mechanism. EMBO J 26:578-588.

15.       Koradi R, Billeter M, & Wuthrich K (1996) MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph 14:51-55, 29-32.

16.       Hayward S & Berendsen HJ (1998) Systematic analysis of domain motions in proteins from conformational change: new results on citrate synthase and T4 lysozyme. Proteins 30:144-154.

17.       Lukin JA & Ho C (2003) Nuclear magnetic resonance of hemoglobins. Methods Mol Med 82:251-269.

18.       Poulos TL (2006) Soluble guanylate cyclase. Curr Opin Struct Biol 16:736-743.

19.       Barrick D (1994) Replacement of the proximal ligand of sperm whale myoglobin with free imidazole in the mutant His-93–>Gly. Biochemistry 33:6546-6554.

20.       Zhao Y, Schelvis JP, Babcock GT, & Marletta MA (1998) Identification of histidine 105 in the beta1 subunit of soluble guanylate cyclase as the heme proximal ligand. Biochemistry 37:4502-4509.

21.       Ferentz AE & Wagner G (2000) NMR spectroscopy: a multifaceted approach to macromolecular structure. Q Rev Biophys 33:29-65.

22.       Lamar GN, Budd DL, Viscio DB, Smith KM, & Langry KC (1978) Proton nuclear magnetic-resonance characterization of heme disorder in hemoproteins. Proc Natl Acad Sci U S A 75:5755-5759.

23.       Powers L, Sessler JL, Woolery GL, & Chance B (1984) CO bond angle changes in photolysis of carboxymyoglobin. Biochemistry 23:5519-5523.

24.       Peng SM & Ibers JA (1976) Stereochemistry of carbonylmetalloporphyrins. The structure of (pyridine)(carbonyl)(5, 10, 15, 20-tetraphenylprophinato)iron(II). Journal of the American Chemical Society 98:8032-8036.

25.       Ivanov D, et al. (1994) Determination of CO orientation in myoglobin by single-crystal infrared linear dichroism. Journal of the American Chemical Society 116:4139-4140.

26.       Ray GB, Li XY, Ibers JA, Sessler JL, & Spiro TG (1994) How far can proteins bend the FeCO unit – distal polar and steric effects in heme-proteins and models. Journal of the American Chemical Society 116:162-176.

27.       Quillin ML, Arduini RM, Olson JS, & Phillips GN (1993) High-resolution crystal-structures of distal histidine mutants of sperm whale myoglobin. Journal of Molecular Biology 234:140-155.

28.       Clore GM & Garrett DS (1999) R-factor, free R, and complete cross-validation for dipolar coupling refinement of NMR structures. Journal of the American Chemical Society 121:9008-9012.

29.       Wei Y & Werner MH (2006) iDC: A comprehensive toolkit for the analysis of residual dipolar couplings for macromolecular structure determination. J Biomol NMR 35:17-25.

30.       Bhattacharya A, Tejero R, & Montelione GT (2007) Evaluating protein structures determined by structural genomics consortia. Proteins 66:778-795.

31.       Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, & Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8:477-486.

32.       Sippl MJ (1993) Recognition of errors in three-dimensional structures of proteins. Proteins 17:355-362.

33.       Lovell SC, et al. (2003) Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins 50:437-450.

34.       Chou JJ, Li S, & Bax A (2000) Study of conformational rearrangement and refinement of structural homology models by the use of heteronuclear dipolar couplings. J Biomol NMR 18:217-227.

35.       Osapay K, Theriault Y, Wright PE, & Case DA (1994) Solution structure of carbonmonoxy myoglobin determined from nuclear magnetic resonance distance and chemical shift constraints. J Mol Biol 244:183-197.

36.       Cross KJ & Wright PE (1985) Calibration of ring-current models for the heme ring. J Magn Reson 64:220-231.

37.       Rivera M & Walker FA (1995) Biosynthetic preparation of isotopically labeled heme. Anal Biochem 230:295-302.

38.       Alontaga AY, Bunce RA, Wilks A, & Rivera M (2006) 13C NMR spectroscopy of core heme carbons as a simple tool to elucidate the coordination state of ferric high-spin heme proteins. Inorg Chem 45:8876-8881.

39.       Stone JR & Marletta MA (1994) Soluble guanylate cyclase from bovine lung: activation with nitric oxide and carbon monoxide and spectral characterization of the ferrous and ferric states. Biochemistry 33:5636-5640.

40.       Perutz MF (1979) Regulation of oxygen affinity of hemoglobin: influence of structure of the globin on the heme iron. Annu Rev Biochem 48:327-386.

41.       Karow DS, et al. (2004) Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and Thermoanaerobacter tengcongensis. Biochemistry 43:10203-10211.

42.       Tolman JR, Flanagan JM, Kennedy MA, & Prestegard JH (1995) Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution. Proc Natl Acad Sci U S A 92:9279-9283.

43.       Tjandra N & Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278:1111-1114.

44.       Murphy EC, Zhurkin VB, Louis JM, Cornilescu G, & Clore GM (2001) Structural basis for SRY-dependent 46-X,Y sex reversal: modulation of DNA bending by a naturally occurring point mutation. J Mol Biol 312:481-499.

45.       Lange OF, et al. (2008) Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 320:1471-1475.

46.       Decatur SM, et al. (1996) Trans effects in nitric oxide binding to myoglobin cavity mutant H93G. Biochemistry 35:4939-4944.

 

 

 

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About kayaerbil

I am a Berkeley educated chemistry Ph.D. who is moving into the area of working on developing appropriate technology for communities that are subjected to socio-economic oppression. The goal is to use simple and effective designs to empower people to live better lives. Currently, I am working with Native Americans on Pine Ridge, the Lakota reservation in South Dakota. I am working with a Native owned and run solar energy company. We are currently working on building a compressed earth block (CEB) house that showcases many of the technologies that the company has developed. The CEB house is made of locally derived resources, earth from the reservation. The blocks are naturally thermally insulating, keeping the house cool in the summer and warm in the winter. Eventually, a solar air heater and photovoltaic panels will be installed into the house to power the home and keep it warm, while preserving the house off the grid. A side project while in Pine Ridge is a solar computer. I hope to learn about blockchain encryption software for building microgrids. In addition, it is an immediate interest of mine to involve local youth in technology education.
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