Investigation of structural and functional properties of the betaine transporter BetP from Corynebacterium glutamicum by using infrared spectroscopy
Başlık çevirisi mevcut değil.
- Tez No: 401193
- Danışmanlar: PROF. DR. WERNER MANTELE
- Tez Türü: Doktora
- Konular: Fizik ve Fizik Mühendisliği, Physics and Physics Engineering
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2013
- Dil: İngilizce
- Üniversite: Johann-Wolfgang-Goethe-Universität
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 179
Özet
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Özet (Çeviri)
In this study, the structural and functional properties of the Na+/Betaine symporter BetP were investigated upon K+-induced activation. BetP regulates transport activity dependent on the amount of associated anionic lipids and the cytoplasmic K+-concentration. For this purpose, FTIR spectroscopy was implemented as a non-perturbing biophysical method which shed light on how the membrane lipids contribute to the molecular mechanisms of activation and regulatory response of BetP. Secondary structure analysis: The FTIR data showed that all forms of BetP (i.e., detergent solubilized, proteoliposomes and 2D crystals) exhibit more or less identical spectra and are mainly α-helical. BetP exhibits 75% α-helix, 12% solvated loops/short helices, 310-helix and/or unordered structure, and 13% restricted turns/loops under inactivating buffer conditions (i.e. without K+). However, the fraction of α-helices decreases by 7% while the amount of restricted turn/loops and solvated/short structures increases by 5% and 2%, respectively, upon activation (with 500 mM K+). These small changes in the fraction of secondary structures of BetP 2D crystals could result from either an alteration in the solvation status of the secondary structures due to a conformational change or from the reorganization of the secondary structures within the protein upon K+-induced activation. K+ titration experiments and activation/deactivation of BetP: The IR data revealed in situ structural changes of BetP WT 2D crystals upon a stepwise activation with increasing K+ ions (0500 mM). On the basis of the FTIR spectra, we observed only slight band shifts for the major amide I components (α-helix and turn/loops) with increasing K+. This suggests that the H-bonding strength of the regular secondary structures is altered under high K+. Observation of small amide I difference signals both in H2O and D2O buffer environment confirms minute conformational alterations in the protein backbone upon upregulation of BetP. 4. SUMMARY 136 On the other hand, several distinct band shifts were detected with increasing K+ above 1700 cm-1 which corresponds to the C=O stretching modes of carboxylate groups of protonated Asp/Glu residues. Strongly, intermediately and weakly H-bonded protonated Asp/Glu respond to K+ increment in a similar manner that the shifts are reversed above ~250 mM K+. Additionally, the signals of protonated Asp/Glu residues intensify upon activation, indicating increase interactions of these residues upon activation. This reflects that the H-bonding properties of protonated Asp/Glu residues are altered; H-bonds are strengthened or weakened/lost upon K+-stimulated activation. Moreover, the C=O stretching modes of carboxylate groups of deprotonated Asp/Glu residues undergo clear spectral shifts concomitant with a decrease in their population in the presence of high K+ concentrations. Thereby, we can conclude that a H-bonded network of both protonated and deprotonated Asp/Glu residues in BetP is disturbed with increasing K+ concentration both in H2O and D2O buffer. Altogether this suggests that eventually a protonation/deprotonation of Asp/Glu residues may be involved in the K+-induced activation of BetP. Unlike wild type BetP, the C-terminal truncated BetP in 2D crystals exhibit only tiny IR-difference signals for protonated Asp/Glu residues above 1700 cm-1 under activating conditions. The IR signals of protonated Asp/Glu and of Arg/Lys residues disappear in the case of this mutant. This indicates that these charged residues respond to an increase of the concentration of K+ only in the case of BetP WT. This clearly proves an important role of the C-terminal domain in the ionic interaction network on the cytoplasmic side of BetP and in the activation/regulation cycle of BetP. It is conceiving that K+-induced activation of BetP involves the interactions of Asp/Glu and Arg residues in the cytoplasmic region. In this respect, the intensity of the IR signals of arginine residues increases dramatically with elevated K+ indicating an increase in the interactions of Arg residues of BetP under activating conditions. Recently, the ionic interaction of terminal domains with cytoplasmic loops via charged residues has been described27. The IR-difference spectra also showed that the C=O modes of Asn and Gln residues intensify significantly concomitant with a shifting of the NH2 bending modes of Asn residues that undergo a change in the H-bonding pattern upon activation. These results are in line with the fact that formation of inter-protomer H-bonds includes Asn (h7) and Thr (loop 7) residues in the interfacial region of BetP. It is likely that conformational alterations occur around Asn/Gln residues on the periplasmic side of the protein. A clear response of the C-C ring mode of tyrosine residues to K+ titration provided dynamic information on changes in the interaction network of BetP during regulation. This is in good agreement with the fact that some tyrosine residues are involved in K+ coordination revealed by structural studies. The IR signals of both protonated and deprotonated Tyr residues exhibit opposite spectral alterations in terms of intensity changes as well as band shifting (a weakening/loss or strengthening of H-bonding) with elevated K+. This suggests that some Tyr residues either play a role in the regulation of BetP activation or sense the movement of the backbone due to conformational alterations in the result of K+ binding. Particularly, the signals of protonated Tyr exhibit a broad band in the IR-difference spectrum (active-minus-inactive), suggesting that a few Tyr residues are involved in the activation of BetP as being reporter and/or actor. It is likely to speculate that residues involved in Rb+ binding or in formation of the periplasmic betaine-binding site (S2 site) might contribute to the observed IR shifts. Besides, the K+-dependent formation of the S2 site occurs through conformational changes in TM12, establishing an additional salt bridge between Tyr206 and Aps547 in the cytoplasmic tip of TM12. The FTIR data of BetP proteoliposomes are in good agreement with 2D crystals. Both forms of BetP exhibit identical spectral alterations, particularly in the protonation states and in the environment of Asp/Glu and Arg residues under high K+. Overall, the FTIR data indicate that loops and solvated helices (C-terminal domain and/or short helices) in the hydrophilic region of BetP, which contain Asp/Glu and Arg residues, are involved in the activation. Asp/Glu residues interact most likely with Arg/Lys residues via salt bridges, being involved in K+-dependent activation of BetP, and the strength of these interactions is also influenced with K+. Observation of tiny difference signals for Trp residues is indicative for reorientations of these residues in response to K+ increase. Altogether this suggests that K+-stimulated activation of BetP is initiated by a H-bonded network of Asp/Glu and Arg residues. pH titration experiments: In situ titration of BetP at various pH values showed the protonation states of Asp/Glu residues with their locations in BetP and the pH-induced conformational changes, sheding light on the ionic interaction networks of terminal domains with cytoplasmic loops via charged residues. According to the FTIR data, the most pronounced spectral changes are monitored in the pH range of 5.5-7.5. The IR signals for protonated Asp/Glu intensify while the population of deprotonated Asp/Glu residues decreases. This suggests that protonation/deprotonation states of Asp/Glu residues are altered in this pH range concomitant with conformational changes of turn/loops as well as with microenviromental changes around Tyr in BetP. Hereby, the pKa value of Asp/Glu residues lies in a wide range of pH5.5-pH7.5 which is remarkably high for a carboxylic acid. This suggests that Asp/Glu residues influenced by pH changes locate most likely away from the solvent and/or these groups make electrostatic interactions with the environment, most likely with some positively charged residues (i.e. Arg/Lys). It was already argued that upon K+-induced activation, both protonated and deprotonated Asp/Glu residues undergo H-bonding changes, and interactions of both Arg and protonated Asp/Glu residues increase under high K+. It is likely that the same groups of Asp/Glu residues are involved in the regulatory activation mechanism of BetP. Lipid-protein interactions: Probing of the lipid signals provided experimental evidence on how BetP dynamically interacts with the bilayer lipids under high K+. Phospholipid headgroups undergo K+ dependent changes in their electrostatic and H-bonding properties favoring weak/free H-bonds, while the hydrophobic tails become more mobile and even disordered upon K+-induced activation of BetP. FTIR data suggest that the inactive state of BetP is characterized by tight lipid-protein interactions most likely similar to what was observed in the latest structure of BetP95. The FTIR data also reveal that K+ abolishes a tight interaction of BetP especially with the anionic lipid headgroups suggesting that lipid-protein interactions have to soften so that BetP can adopt its active state. Hereby, the hypothesized molecular switch model was confirmed that due to K+ activation the positively charged C-terminal domains switch their interaction partner from lipids (under down-regulating conditions) to protein-interactions, which could also involve intratrimeric interactions between C-terminal domains as suggested by structural studies and the fact that monomeric BetP is down-regulated. 1H/2H exchange: 1H/2H exchange experiments provided information regarding the solvent accessibility in the open and closed states of BetP. On the basis of the 1H/2H exchange extent and kinetics, the fraction of the exchanged amide groups is significantly larger in the active state (58%) than in the inactive state (52%) at the end of 3 hours deuteration, indicating a more open conformation of BetP WT 2D crystals upon a rise in the K+ concentration. Interestingly, inactive BetP exhibits faster exchange of amide protons in the first 25 min than the active BetP, reflecting abundance of unordered structures, loops and solvated helices in the absence of K+. A similar H/D exchange profile was observed in the case of proteoliposomes of BetP. This suggests that some solvated loops and/or short helices located in the hydrophilic region of BetP make additional interactions, hiding somewhere and being away from the solvent upon activation. The FTIR data also reveal that Asp/Glu and Arg residues, located in loops and in the C-terminal domain, are deuterated further with increasing K+ concentration. It is likely that these residues change their interaction partners shielding from the solvent in the result of reorientation of the C-terminal domain as well as solvated loops upon activation. Thermal stability: Both in the active and inactive sates of BetP WT 2D crystals, loops/turns and 310-helix unfold with appearing of the intermolecular β-sheet signals, being affecting by temperature changes prior to α-helices. α-helix and turns/loops of active BetP exhibit a Tm of 79°C and 77°C, respectively, while they are 76°C and 74°C for inactive BetP. Moreover, both α-helix and turns/loops exhibit a 3°C higher Tm under high K+. This proves that active BetP has more rigid loops and helices as compared to inactive BetP, reflecting a more stable status of BetP in the active state with K+ ions. This findings are in perfect agreement with the H/D exchange results as well as with the thermal stability analysis of BetP WT proteoliposomes. Unlike BetP WT 2D crystals, the C-terminal domain truncated BetP 2D crystals is also affected by rising temperature in a similar manner but without significant differences between the both states, indicating different interactions of the C-terminal domain at the active and inactive states of BetP. In summary, probing of the lipid-protein interactions as well as K+-dependent structural changes provided more holistic information on function-related structural properties of BetP. Altogether the FTIR data suggest that secondary structure rearrangements and observation of interactions between charged residues are the 'hot spots' which are involved in the activation cycle of BetP. It can be postulated that negatively charged phosphate lipid headgroups are released from the positively charged Arg residues located at the end of the C-terminal domain upon K+-binding and might undergo a change in interaction towards loops or the N-terminal domains. This is followed by reorientation of C-termini/transmembrane helices and by unzipping of interactions of charged residues between the cytoplasmic loops and C-termini, resulting in structural opening in the active state of BetP. Hereby, new insights into the structure-function relationship of BetP upon activation selectively induced by K+ ions were revealed on the molecular level with IR spectroscopy.
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