Regulation of Acetate Kinase Isozymes and Its Importance for MixedAcid Fermentation in Lactococcus lactis
Puri, P. ; Goel, A. ; Bochynska, A. ; Poolman, B. - \ 2014
Journal of Bacteriology 196 (2014)7. - ISSN 0021-9193 - p. 1386 - 1393.
streptococcus-lactis - escherichia-coli - methanosarcina-thermophila - lysine acetylation - membrane-proteins - product formation - light-scattering - in-vivo - metabolism - phosphate
Acetate kinase (ACK) converts acetyl phosphate to acetate along with the generation of ATP in the pathway for mixed-acid fermentation in Lactococcus lactis. The reverse reaction yields acetyl phosphate for assimilation purposes. Remarkably, L. lactis has two ACK isozymes, and the corresponding genes are present in an operon. We purified both enzymes (AckA1 and AckA2) from L. lactis MG1363 and determined their oligomeric state, specific activities, and allosteric regulation. Both proteins form homodimeric complexes, as shown by size exclusion chromatography and static light-scattering measurements. The turnover number of AckA1 is about an order of magnitude higher than that of AckA2 for the reaction in either direction. The K-m values for acetyl phosphate, ATP, and ADP are similar for both enzymes. However, AckA2 has a higher affinity for acetate than does AckA1, suggesting an important role under acetate-limiting conditions despite the lower activity. Fructose-1,6-bisphosphate, glyceraldehyde- 3-phosphate, and phospho-enol-pyruvate inhibit the activities of AckA1 and AckA2 to different extents. The allosteric regulation of AckA1 and AckA2 and the pool sizes of the glycolytic intermediates are consistent with a switch from homolactic to mixed-acid fermentation upon slowing of the growth rate.
Pathway transfer in fungi: Transporters are the key to success
Straat, L. van der; Graaff, L.H. de - \ 2014
Bioengineered 5 (2014)5. - ISSN 2165-5979 - p. 335 - 339.
itaconic acid production - saccharomyces-cerevisiae - lactococcus-lactis - aspergillus-niger - cephalosporin production - penicillium-chrysogenum - functional expression - membrane-proteins - ceft gene - overproduction
Itaconic acid is an important building block for the chemical industry. Currently, Aspergillus terreus is the main organism used for itaconic acid production. Due to the enormous citric acid production capacity of Aspergillus niger, this host is investigated as a potential itaconic acid production host. Several strategies have been tried so far: fermentation optimization, expression of cis-aconitate decarboxylase (cadA) alone and in combination with aconitase targeted to the same compartment, chassis optimization, and the heterologous expression of two transporters flanking the cadA gene. We showed that the heterologous expression of these two transporters were key to improving itaconic acid production in an A. niger strain that was unable to produce oxalic acid and gluconic acid. The expression of transporters has increased the production levels of other industrially relevant processes as well, such as ß-lactam antibiotics and bioethanol. Thus far, the role of transporters in production process optimization is a bit overlooked.
Arabidopsis COBRA-LIKE 10, a GPI-anchored protein, mediates directional growth of pollen tubes
Li, S. ; Ge, F.R. ; Xu, M. ; Zhao, X.Y. ; Huang, G.Q. ; Zhou, L.Z. ; Wang, J.G. ; Kombrink, A. ; McCormick, S. ; Zhang, X.S. ; Zhang, Y. - \ 2013
The Plant Journal 74 (2013)3. - ISSN 0960-7412 - p. 486 - 497.
male gametophyte development - oriented cell expansion - membrane-proteins - polarized growth - tip growth - in-vitro - thaliana - gene - guidance - encodes
Successful reproduction of flowering plants requires constant communication between female tissues and growing pollen tubes. Female cells secrete molecules and peptides as nutrients or guidance cues for fast and directional tube growth, which is executed by dynamic changes of intracellular activities within pollen tubes. Compared with the extensive interest in female cues and intracellular activities of pollen tubes, how female cues are sensed and interpreted intracellularly in pollen is poorly understood. We show here that COBL10, a glycosylphosphatidylinositol (GPI)-anchored protein, is one component of this pollen tube internal machinery. Mutations in COBL10 caused gametophytic male sterility due to reduced pollen tube growth and compromised directional sensing in the female transmitting tract. Deposition of the apical pectin cap and cellulose microfibrils was disrupted in cobl10 pollen tubes. Pollen tube localization of COBL10 at the apical plasma membrane is critical for its function and relies on proper GPI processing and its C-terminal hydrophobic residues. GPI-anchored proteins are widespread cell sensors in mammals, especially during egg-sperm communication. Our results that COBL10 is critical for directional growth of pollen tubes suggest that they play critical roles in cell-cell communications in plants.
SDSL-ESR-based protein structure characterization
Strancar, J. ; Kavalenka, A.A. ; Urbancic, I. ; Ljubetic, A. ; Hemminga, M.A. - \ 2010
European Biophysics Journal 39 (2010)4. - ISSN 0175-7571 - p. 499 - 511.
intrinsically unstructured proteins - molecular-dynamics simulations - natively unfolded proteins - side-chain conformation - small-angle scattering - membrane-proteins - biosystem complexity - pancreatic lipase - crystal-structure - epr spectroscopy
As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structure
Conformational studies of peptides representing a segment of TM7 from Vo-H+-V-ATPase in SDS micelles
Duarte, A.M. ; Jong, E.R. de; Koehorst, R.B.M. ; Hemminga, M.A. - \ 2010
European Biophysics Journal 39 (2010)4. - ISSN 0175-7571 - p. 639 - 646.
major coat protein - proton translocation channel - 5th transmembrane segment - dodecyl-sulfate micelles - membrane-proteins - escherichia-coli - nmr - bacteriophage-m13 - mimicking - detergent
The conformation of a transmembrane peptide, sMTM7, encompassing the cytoplasmic hemi-channel domain of the seventh transmembrane section of subunit a from V-ATPase from Saccharomyces cerevisiae solubilized in SDS solutions was studied by circular dichroism (CD) spectroscopy and fluorescence spectroscopy of the single tryptophan residue of this peptide. The results show that the peptide adopts an alpha-helical conformation or aggregated beta-sheet depending on the peptide-to-SDS ratio used. The results are compared with published data about a longer version of the peptide (i.e., MTM7). It is concluded that the bulky, positively charged arginine residue located in the center of both peptides has a destabilizing effect on the helical conformation of the SDS-solubilized peptides, leading to beta-sheet formation and subsequent aggregation.
Asymmetric dipping of bacteriophage M13 coat protein with increasing lipid bilayer thickness
Stopar, D. ; Koehorst, R.B.M. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2009
Biochimica et Biophysica Acta. Biomembranes 1788 (2009)10. - ISSN 0005-2736 - p. 2217 - 2221.
membrane-proteins - tryptophan residues - amino-acids - peptides - topology - helix - spectroscopy - orientation
Knowledge about the vertical movement of a protein with respect to the lipid bilayer plane is important to understand protein functionality in the biological membrane. In this work, the vertical displacement of bacteriophage M13 major coat protein in a lipid bilayer is used as a model system to study the molecular details of its anchoring mechanism in a homologue series of lipids with the same polar head group but different hydrophobic chain length. The major coat proteins were reconstituted into 14:1PC, 16:1PC, 18:1PC, 20:1PC, and 22:1PC bilayers, and the fluorescence spectra were measured of the intrinsic tryptophan at position 26 and BADAN attached to an introduced cysteine at position 46, located at the opposite ends of the transmembrane helix. The fluorescence maximum of tryptophan shifted for 700 cm-1 on going from 14:1PC to 22:1PC, the corresponding shift of the fluorescence maximum of BADAN at position 46 was approximately 10 times less ( 70 cm-1). Quenching of fluorescence with the spin label CAT 1 indicates that the tryptophan is becoming progressively inaccessible for the quencher with increasing bilayer thickness, whereas quenching of BADAN attached to the T46C mutant remained approximately unchanged. This supports the idea that the BADAN probe at position 46 remains at the same depth in the bilayer irrespective of its thickness and clearly indicates an asymmetrical nature of the protein dipping in the lipid bilayer. The anchoring strength at the C-terminal domain of the protein (provided by two phenylalanine residues together with four lysine residues) was estimated to be roughly 5 times larger than the anchoring strength of the N-terminal domain
Tilt and Rotation Angles of a Transmembrane Model Peptide as Studied by Fluorescence Spectroscopy
Holt, A. ; Koehorst, R.B.M. ; Rutters-Meijeke, T. ; Gelb, M.H. ; Rijkers, D.T.S. ; Hemminga, M.A. ; Killian, J.A. - \ 2009
Biophysical Journal 97 (2009)8. - ISSN 0006-3495 - p. 2258 - 2266.
molecular-dynamics simulations - alpha-helical peptides - major coat protein - solid-state nmr - lipid-bilayers - hydrophobic mismatch - membrane-proteins - geometric analysis - aromatic residues - self-association
In this study the membrane orientation of a tryptophan-flanked model peptide, WALP23, was determined by using peptides that were labeled at different positions along the sequence with the environmentally sensitive fluorescent label BADAN. The fluorescence properties, reflecting the local polarity, were used to determine the tilt and rotation angles of the peptide based on an ideal a-helix model. For WALP23 inserted in dioleoylphosphatidylcholine (DOPC), an estimated tilt angle of the helix with respect to the bilayer normal of 24° ± 5° was obtained. When the peptides were inserted into bilayers with different acyl chain lengths or containing different concentrations of cholesterol, small changes in tilt angle were observed as response to hydrophobic mismatch, whereas the rotation angle appeared to be independent of lipid composition. In all cases, the tilt angles were significantly larger than those previously determined from 2H NMR experiments, supporting recent suggestions that the relatively long timescale of 2H NMR measurements may result in an underestimation of tilt angles due to partial motional averaging. It is concluded that although the fluorescence technique has a rather low resolution and limited accuracy, it can be used to resolve the discrepancies observed between previous 2H NMR experiments and molecular-dynamics simulations
Site-directed spin labeling study of the light-harvesting complex CP29
Kavalenka, A.A. ; Spruijt, R.B. ; Wolfs, C.J.A.M. ; Strancar, J. ; Croce, R. ; Hemminga, M.A. ; Amerongen, H. van - \ 2009
Biophysical Journal 96 (2009)9. - ISSN 0006-3495 - p. 3620 - 3628.
chlorophyll-a/b protein - photosystem-ii subunit - plant antenna protein - n-terminal domain - energy-transfer - conformational-changes - biosystem complexity - membrane-proteins - escherichia-coli - binding protein
The topology of the long N-terminal domain (100 amino-acid residues) of the photosynthetic Lhc CP29 was studied using electron spin resonance. Wild-type protein containing a single cysteine at position 108 and nine single-cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases, the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. The spin-label electron spin resonance spectra were analyzed in terms of a multicomponent spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. These results permit to trace the structural organization of the long N-terminal domain of CP29. Amino-acid residues 97 and 108 are located in the transmembrane pigment-containing protein body of the protein. Positions 65, 81, and 90 are located in a flexible loop that is proposed to extend out of the protein from the stromal surface. This loop also contains a phosphorylation site at Thr81, suggesting that the flexibility of this loop might play a role in the regulatory mechanisms of the light-harvesting process. Positions 4, 33, 40, and 56 are found to be located in a relatively rigid environment, close to the transmembrane protein body. On the other hand, position 15 is located in a flexible region, relatively far away from the transmembrane domain
Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-ATPase
Duarte, A.M. ; Wolfs, C.J.A.M. ; Nuland, N.A.J. van; Harrison, M.A. ; Findlay, J.B.C. ; Mierlo, C.P.M. van; Hemminga, M.A. - \ 2007
Biochimica et Biophysica Acta. Biomembranes 1768 (2007)2. - ISSN 0005-2736 - p. 218 - 227.
nuclear-magnetic-resonance - sarcoplasmic-reticulum ca2+-atpase - protein secondary structure - circular-dichroism spectra - sodium dodecyl-sulfate - m13 coat protein - v-atpase - vacuolar (h+)-atpases - membrane-proteins - nmr-spectroscopy
Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an ¿-helical conformation for peptide MTM7 and in DMSO three ¿-helical regions are identified by 2D 1H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an ¿-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase
Potato tuber proteomics: Comparison of two complementary extraction methods designed for 2-DE of acidic proteins
Delaplace, P. ; Wal, F. van der; Dierick, J.F. ; Cordewener, J.H.G. ; Fauconnier, M.L. ; Jardin, P. du; America, A.H.P. - \ 2006
Proteomics 6 (2006)24. - ISSN 1615-9853 - p. 6494 - 6497.
dimensional gel-electrophoresis - recalcitrant plant-tissues - 2-dimensional electrophoresis - membrane-proteins - solanum-tuberosum - solubilization - dormancy - storage
Two protein extraction procedures were tested in order to remove interfering compounds prior to 2-DE of potato tubers. These methods using SDS lysis buffer and phenol-phase extraction were compared regarding the quality of the resulting 2-D gel. While the resolution of SDS extracts on semipreparative gels seems better, both methods lead to similar extraction yields and total number of spots. The procedures are complementary regarding the Mr range of preferentially extracted proteins
Alignment and statistical difference analysis of complex peptide data sets generated by multidimensional LC-MS
America, A.H.P. ; Cordewener, J.H.G. ; Geffen, M.H.A. van; Lommen, A. ; Vissers, J.P.C. ; Bino, R.J. ; Hall, R.D. - \ 2006
Proteomics 6 (2006)2. - ISSN 1615-9853 - p. 641 - 653.
mass-spectrometry - liquid-chromatography - comparative proteomics - gel-electrophoresis - protein expression - membrane-proteins - metabolites - technology - mixtures
A method for high-resolution proteomics analyses of complex protein mixtures is presented using multidimensional HPLC coupled to MS (MDLC-MS). The method was applied to identify proteins that are differentially expressed during fruit ripening of tomato. Protein extracts from red and green tomato fruits were digested by trypsin. The resulting highly complex peptide mixtures were separated by strong cation exchange chromatography (SCX), and subsequently analyzed by RP nano-LC coupled to quadrupole-TOF MS. For detailed quantitative comparison, triplicate RP-LC-MS runs were performed for each SCX fraction. The resulting data sets were analyzed using MetAlign software for noise and data reduction, multiple alignment and statistical variance analysis. For each RP-LC-MS chromatogram, up to 7000 mass components were detected. Peak intensity data were compared by multivariate and statistical analysis. This revealed a clear separation between the green and red tomato samples, and a clear separation of the different SCX fractions. MS/MS spectra were collected using the data-dependent acquisition mode from a selected set of differentially detected peptide masses, enabling the identification of proteins that were differentially expressed during ripening of tomato fruits. Our approach is a highly sensitive method to analyze proteins in complex mixtures without the need of isotope labeling.
Protein-lipid interactions of bacteriophage M13 gene 9 minor coat protein (Review)
Houbiers, M.C. ; Hemminga, M.A. - \ 2004
Molecular Membrane Biology 21 (2004)6. - ISSN 0968-7688 - p. 351 - 359.
membrane-proteins - escherichia-coli - phage display - alpha-helices - m13 procoat - filamentous bacteriophage-m13 - nmr-spectroscopy - charged residues - outer membranes - inner membrane
Gene 9 protein is one of the minor coat proteins of bacteriophage M13. The protein plays a role in the assembly process by associating with the host membrane by protein-lipid interactions. The availability of chemically synthesized protein has enabled the biophysical characterization of the membrane-bound state of the protein by using model membrane systems. This paper summarizes, discusses and further interprets this work in the light of the current state of the literature, leading to new possible models of the coat protein in a membrane. The biological implications of these findings related to the membrane-bound phage assembly are indicated.
Profiling of the secreted proteins during 3T3-L1 adipocyte differentiation leads to the identification of novel adipokines
Wang, P. ; Mariman, E. ; Keijer, J. ; Noben, J.P. ; Robben, J. ; Renes, J. - \ 2004
Cellular and Molecular Life Sciences 61 (2004)8. - ISSN 1420-682X - p. 2405 - 2417.
inhibitory factor mif - cis-trans-isomerases - adipose-tissue - extracellular-matrix - gene-expression - potential role - insulin-resistance - membrane-proteins - alpha-enolase - growth-factor
Adipose tissue is an endocrine organ capable of secreting a number of adipokines with a role in the regulation of adipose tissue and whole-body metabolism. We used two-dimensional gel electrophoresis combined with mass spectrometry to profile the secreted proteins from (pre)adipocytes. The culture medium of 3T3-L1 cells during adipocyte differentiation was screened, and 41 proteins that responded to blocking of secretion by 20°C treatment and/or brefeldin A treatment were identified. Prohibitin, stress-70 protein, and adhesion-regulating molecule 1 are reported for the first time as secreted proteins. In addition, procollagen C-proteinase enhancer protein, galectin-1, cyclophilin A and C, and SF20/IL-25 are newly identified as adipocyte secreted factors. Secretion profiles indicated a dynamic environment including an actively remodeling extracellular matrix and several factors involved in growth regulation