Richard Needleman

Glutamic Acid 204 is the Terminal Proton Release Group at the Extracellular Surface of Bacteriorhodopsin

We have measured proton release into the medium after proton transfer from the retinal Schiff base to Asp85 in the photocycle and the C=O stretch bands of carboxylic acids in wild type bacteriorhodopsin and the E204Q and E204D mutants. In E204Q, but not in E204D, the normal proton release is absent. Consistent with this, a negative band in the Fourier transform infrared difference spectra at 1700 cm−1 in the wild type, which we now attribute to depletion of the protonated E204, is also absent in E204Q. In E204D, this band is shifted to 1714 cm−1, as expected from the higher frequency for a protonated aspartic than for a glutamic acid. Consistent with their origin from protonated carboxyls, the depletion bands in the wild type and E204D shift in D2O to 1690 and 1703 cm−1, respectively. In the protein structure, Glu204 seems to be connected to the Schiff base region by a chain of hydrogen-bonded water. As with other residues closer to the Schiff base, replacement of Glu204 with glutamine changes the O-H stretch frequency of the bound water molecule near Asp85 that undergoes hydrogen-bonding change in the photocycle. The results therefore identify Glu204 as XH, the earlier postulated residue that is the source of the released proton during the transport, and suggest that its deprotonation is triggered by the protonation of Asp85 through a network that contains water dipoles.

An efficient system for the synthesis of bacteriorhodopsin in Halobacterium halobium

The mechanism by which bacteriorhodopsin (BR) transports protons across the cell membrane of Halobacterium halobium is actively studied in many laboratories. Currently available systems for the synthesis of mutant proteins obtained by site-directed mutagenesis of the gene encoding BR (bop) require reconstitution of the denatured polypeptide after its synthesis Escherichia coli or yeast; this approach is technically difficult and labor intensive, and raises questions about possible differences between in vivo and in vitro folding. Using a newly described transformation system and a halobacterial plasmid vector, we show that it is possible to reintroduce the bop gene into BR- strains of H. halobium. The bop-carrying plasmid expresses native BR in amounts similar to those obtained in several wild type strains. This system allows facile site-directed mutagenesis in halophilic archaebacteria.

Location of a Cation-Binding Site in the Loop between Helices F and G of Bacteriorhodopsin as Studied by 13C NMR

The high-affinity cation-binding sites of bacteriorhodopsin (bR) were examined by solid-state 13C NMR of samples labeled with [3-13C]Ala and [1-13C]Val. We found that the 13C NMR spectra of two kinds of blue membranes, deionized (pH 4) and acid blue at pH 1.2, were very similar and different from that of the native purple membrane. This suggested that when the surface pH is lowered, either by removal of cations or by lowering the bulk pH, substantial change is induced in the secondary structure of the protein. Partial replacement of the bound cations with Na+, Ca2+, or Mn2+ produced additional spectral changes in the 13C NMR spectra. The following conclusions were made. First, there are high-affinity cation-binding sites in both the extracellular and the cytoplasmic regions, presumably near the surface, and one of the preferred cation-binding sites is located at the loop between the helix F and G (F-G loop) near Ala196, consistent with the 3D structure of bR from x-ray diffraction and cryoelectron microscopy. Second, the bound cations undergo rather rapid exchange (with a lifetime shorter than 3 ms) among various types of cation-binding sites. As expected from the location of one of the binding sites, cation binding induced conformational alteration of the F-G interhelical loop.

Methionine sulfoximine, an inhibitor of glutamine synthetase, lowers brain glutamine and glutamate in a mouse model of ALS

In an effort to alter the levels of neurochemicals involved in excitotoxicity, we treated mice with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. Since glutamate toxicity has been proposed as a mechanism for the degeneration of motor neurons in a variety of neurodegenerative diseases, we tested the effects of MSO on the transgenic mouse that overexpresses the mutant human SOD1(G93A) gene, an animal model for the primary inherited form of the human neurodegenerative disease amyotrophic lateral sclerosis (ALS). This treatment in vivo reduced glutamine synthetase activity measured in vitro by 85%. Proton magnetic resonance spectroscopy, with magic angle spinning of intact samples of brain tissue, showed that MSO treatment reduced brain levels of glutamine by 60% and of glutamate by 30% in both the motor cortex and the anterior striatum, while also affecting levels of GABA and glutathione. Kaplan-Meyer survival analysis revealed that MSO treatment significantly extended the lifespan of these mice by 8% (p<0.01). These results show that in the SOD1(G93A) model of neurodegenerative diseases, the concentration of brain glutamate (determined with (1)H-MRS) can be lowered by inhibiting in vivo the synthesis of glutamine with non-toxic doses of MSO.

Connectivity of the Retinal Schiff Base to Asp85 and Asp96 during the Bacteriorhodopsin Photocycle: The Local-Access Model

In the recently proposed local-access model for proton transfers in the bacteriorhodopsin transport cycle (Brown et al. 1998. Biochemistry. 37:3982-3993), connection between the retinal Schiff base and Asp85 (in the extracellular direction) and Asp96 (in the cytoplasmic direction)is maintained as long as the retinal is in its photoisomerized state. The directionality of the proton translocation is determined by influences in the protein that make Asp85 a proton acceptor and, subsequently, Asp96 a proton donor. The idea of concurrent local access of the Schiff base in the two directions is now put to a test in the photocycle of the D115N/D96N mutant. The kinetics had suggested that there is a single sequence of intermediates, L<-->M1<-->M2<-->N, and the M2-->M1 reaction depends on whether a proton is released to the extracellular surface. This is now confirmed. We find that at pH 5, where proton release does not occur, but not at higher pH, the photostationary state created by illumination with yellow light contains not only the M1 and M2 states, but also the L and the N intermediates. Because the L and M1 states decay rapidly, they can be present only if they are in equilibrium with later intermediates of the photocycle. Perturbation of this mixture with a blue flash caused depletion of the M intermediate, followed by its partial recovery at the expense of the L state. The change in the amplitude of the C=O stretch band at 1759 cm-1 demonstrated protonation of Asp85 in this process. Thus, during the reequilibration the Schiff base lost its proton to Asp85. Because the N state, also present in the mixture, arises by protonation of the Schiff base from the cytoplasmic surface, these results fulfill the expectation that under the conditions tested the extracellular access of the Schiff base would not be lost at the time when there is access in the cytoplasmic direction. Instead, the connectivity of the Schiff base flickers rapidly (with the time constant of the M1<-->M2 equilibration) between the two directions during the entire L-to-N segment of the photocycle.

Conformational change of helix G in the bacteriorhodopsin photocycle: investigation with heavy atom labeling and x-ray diffraction.

According to the current structural model of bacteriorhodopsin, Ile222 is located at the cytoplasmic end of helix G. We labeled the single cysteine of the site-directed mutant Ile222 --> Cys with p-chloromercuribenzoic acid and determined the position of the labeled mercury by x-ray diffraction in the unphotolyzed state, and in the MN photointermediate accumulated in the presence of guanidine hydrochloride at pH 9.5. According to the difference Fourier maps between the MN intermediate and the unphotolyzed state, the structural change in the MN intermediate was not affected by mercury labeling. The difference Fourier map between the labeled and the unlabeled I222C gave the position of the mercury label. This information was obtained for both the unphotolyzed state and the MN intermediate. We found that the position of the mercury at residue 222 is shifted by 2.1 +/- 0.8 A in the MN intermediate. This agrees with earlier results that suggested a structural change in the G helix. The movement of the mercury label is so large that it must originate from a cooperative conformational change in the helix G at its cytoplasmic end, rather than from displacement of residue 222. Because Ile222 is located at the same level on the z coordinate as Asp96, the structural change in the G helix could have the functional role of perturbing the environment and therefore the pKa of this functionally important aspartate.

PURIFICATION AND BINDING PROPERTIES OF THE MAL63P ACTIVATOR OF SACCHAROMYCES CEREVISIAE

Mal63p is a transcriptional activator for maltose fermentation in Saccharomyces cerevisiae. We have purified it to homogeneity from a yeast strain in which the MAL63 gene is under the control of the GAL1–GAL10 promoter. Purification included fractionation of a whole-cell extract by ion-exchange chromatography, chromatography using both non-specific DNA-affinity (calf thymus), and sequence-specific DNA-affinity chromatography. Mal63p activity was assayed by its binding to a fragment of the MAL61–MAL62 promoter, using both filter-binding and electrophoretic-mobility shift assays. DNase-I footprinting identified a new binding site (site 3) between the two previously known sites (sites 1 and 2). Mal63p is a dimer, and methylation-protection experiments identify the recognition motif as: c/a GC N9 c/a GC/g.

Repeated Family of Genes Controlling Maltose Fermentation in Saccharomyces carlsbergensis

Maltose fermentation in Saccharomyces spp. requires the presence of any one of five unlinked genes: MAL1, MAL2, MAL3, MAL4, or MAL6. Although the genes are functionally equivalent, their natures and relationships to each other are not known. At least three proteins are necessary for maltose fermentation: maltase, maltose permease, and a regulatory protein. The MAL genes may code for one or more of these proteins. Recently a DNA fragment containing a maltase structural gene has been cloned from a MAL6 strain, CB11, to produce plasmid pMAL9-26. We have conducted genetic and physical analyses of strain CB11. The genetic analysis has demonstrated the presence of two cryptic MAL genes in CB11, MAL1g and MAL3g (linked to MAL1 and to MAL3, respectively), in addition to the MAL6 locus. The physical analysis, which used a subclone of plasmid pMAL9-26 as a probe, detected three HindIII genomic fragments with homology to the probe. Each fragment was shown to be linked to one of the MAL loci genetically demonstrated to be present in CB11. Our results indicate that the cloned maltase structural gene in plasmid pMAL9-26 is linked to MAL6. Since the MAL6 locus has previously been shown to contain a regulatory gene, the MAL6 locus must be a complex locus containing at least two of the factors needed for maltose fermentation: the structural gene for maltase and the maltase regulatory protein. The absence of other fragments which hybridize to the MAL6-derived probe shows that either MAL2 and MAL4 are not related to MAL6, or the DNA corresponding to these genes is absent from the MAL6 strain CB11.

Binding of calcium ions to bacteriorhodopsin.

Adding Ca2+ or other cations to deionized bacteriorhodopsin causes a blue to purple color shift, a result of deprotonation of Asp85. It has been proposed by different groups that the protonation state of Asp85 responds to the binding of Ca2+ either 1) directly at a specific site in the protein or 2) indirectly through the rise of the surface pH. We tested the idea of specific binding of Ca2+ and found that the surface pH, as determined from the ionization state of eosin covalently linked to engineered cysteine residues, rises about equally at both extracellular and cytoplasmic surfaces when only one Ca2+ is added. This precludes binding to a specific site and suggests that rather than decreasing the pKa of Asp85 by direct interaction, Ca2+ increases the surface pH by binding to anionic lipid groups. As Ca2+ is added the surface pH rises, but deprotonation of Asp85 occurs only when the surface pH approaches its pKa. The nonlinear relationship between Ca2+ binding and deprotonation of Asp85 from this effect is different in the wild-type protein and in various mutants and explains the observed complex and varied spectral titration curves.

MAL63 codes for a positive regulator of maltose fermentation in Saccharomyces cerevisiae

SummaryGenetic analysis of the MAL6 locus has previously yielded mal6 mutants which fall into a single complementation group and which are noninducible for maltase and maltose permease. However, the strains used in these studies contained additional partially functional copies of MAL1 (referred to as MAL1g) and MAL3 (referred to as MAL3g). Using a strain lacking MALg genes, we have isolated two classes of mutants and these classes correspond to mutations in MAL63 and MAL61, two genes of the MAL6 complex. Disruptions of MAL63 are noninducible for maltase and maltose permease and for their corresponding mRNAs. The mal6 mutants are shown to map to MAL63 Inducer exclusion as a cause of the noninducible phenotype of the mal63 mutations has been eliminated by constructing a ma163 mutant in a strain constitutive for maltose permease; the strain remains noninducible. These results rigorously demonstrate that MAL63 is a regulatory gene which plays a positive role in the regulation of maltose fermentation.