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1.
Trevino SR Gokulan K Newsom S Thurlkill RL Shaw KL Mitkevich VA Makarov AA Sacchettini JC Scholtz JM Pace CN 《Journal of molecular biology》2005,354(4):967-978
The two most buried carboxyl groups in ribonuclease Sa (RNase Sa) are Asp33 (99% buried; pK 2.4) and Asp79 (85% buried; pK 7.4). Above these pK values, the stability of the D33A variant is 6kcal/mol less than wild-type RNase Sa, and the stability of the D79A variant is 3.3kcal/mol greater than wild-type RNase Sa. The key structural difference between the carboxyl groups is that Asp33 forms three intramolecular hydrogen bonds, and Asp79 forms no intramolecular hydrogen bond. Here, we focus on Asp79 and describe studies of 11 Asp79 variants. Most of the variants were at least 2kcal/mol more stable than wild-type RNase Sa, and the most interesting was D79F. At pH 3, below the pK of Asp79, RNase Sa is 0.3kcal/mol more stable than the D79F variant. At pH 8.5, above the pK of Asp79, RNase Sa is 3.7kcal/mol less stable than the D79F variant. The unfavorable contribution of Asp79 to the stability appears to result from the Born self-energy of burying the charge and, more importantly, from unfavorable charge-charge interactions. To counteract the effect of the negative charge on Asp79, we prepared the Q94K variant and the crystal structure showed that the amino group of the Lys formed a hydrogen-bonded ion pair (distance, 2.71A; angle, 100 degrees ) with the carboxyl group of Asp79. The stability of the Q94K variant was about the same as the wild-type at pH 3, where Asp79 is uncharged, but 1kcal/mol greater than that of wild-type RNase Sa at pH 8.5, where Asp79 is charged. Differences in hydrophobicity, steric strain, Born self-energy, and electrostatic interactions all appear to contribute to the range of stabilities observed in the variants. When it is possible, replacing buried, non-hydrogen bonded, ionizable side-chains with non-polar side-chains is an excellent means of increasing protein stability. 相似文献
2.
Thurlkill RL Grimsley GR Scholtz JM Pace CN 《Protein science : a publication of the Protein Society》2006,15(5):1214-1218
We have used potentiometric titrations to measure the pK values of the ionizable groups of proteins in alanine pentapeptides with appropriately blocked termini. These pentapeptides provide an improved model for the pK values of the ionizable groups in proteins. Our pK values determined in 0.1 M KCl at 25 degrees C are: 3.67+/-0.03 (alpha-carboxyl), 3.67+/-0.04 (Asp), 4.25+/-0.05 (Glu), 6.54+/-0.04 (His), 8.00+/-0.03 (alpha-amino), 8.55+/-0.03 (Cys), 9.84+/-0.11 (Tyr), and 10.40+/-0.08 (Lys). The pK values of some groups differ from the Nozaki and Tanford (N & T) pK values often used in the literature: Asp (3.67 this work vs. 4.0 N & T); His (6.54 this work vs. 6.3 N & T); alpha-amino (8.00 this work vs. 7.5 N & T); Cys (8.55 this work vs. 9.5 N & T); and Tyr (9.84 this work vs. 9.6 N & T). Our pK values will be useful to those who study pK perturbations in folded and unfolded proteins, and to those who use theory to gain a better understanding of the factors that determine the pK values of the ionizable groups of proteins. 相似文献
3.
Gerald R. Grimsley J. Martin Scholtz C. Nick Pace 《Protein science : a publication of the Protein Society》2009,18(1):247-251
We tabulated 541 measured pK values reported in the literature for the Asp, Glu, His, Cys, Tyr, and Lys side chains, and the C and N termini of 78 folded proteins. The majority of these values are for the Asp, Glu, and His side chains. The average pK values are Asp 3.5 ± 1.2 (139); Glu 4.2 ± 0.9 (153); His 6.6 ± 1.0 (131); Cys 6.8 ± 2.7 (25); Tyr 10.3 ± 1.2 (20); Lys 10.5 ± 1.1 (35); C‐terminus 3.3 ± 0.8 (22) and N‐terminus 7.7 ± 0.5 (16). We compare these results with the measured pK values of these groups in alanine pentapeptides, and comment on our overall findings. 相似文献
4.
The energetic consequences of site-specific replacement of a residue at a partially buried site in the two homologous HPr proteins from Escherichia coli and Bacillus subtilis is described. We determined previously that the replacement of a partially buried Lys residue with Glu at position 49 in E.coli HPr increased the conformational stability of the protein substantially because the side-chain of the latter residue could act as a hydrogen-bond acceptor. Here, we extend this analysis to other side-chains with different chemical properties and abilities to form hydrogen bonds to compare the properties of this position in the backgrounds of two different homologous HPr proteins. We find that the variants with polar residues that can form a tertiary hydrogen bond with a nearby site in the protein are more stable than either hydrophobic residues or polar residues that become buried yet are incapable of forming a new hydrogen bond. Furthermore, the protein with the wild-type residue in each HPr variant is not among the most stable of the proteins studied. These results suggest a general strategy for designing variants in which the overall stability of a protein can be modulated in a defined fashion. 相似文献
5.
Experimentally determined mean pK(a) values of carboxyl residues located at the N-termini of alpha-helices are lower than their overall mean values. Here, we perform three types of analyses to account for this phenomenon. We estimate the magnitude of the helix macrodipole to determine its potential role in lowering carboxyl pK(a) values at the N-termini. No correlation between the magnitude of the macrodipole and the pK(a) values is observed. Using the pK(a) program propKa we compare the molecular surroundings of 18 N-termini carboxyl residues versus 233 protein carboxyl groups from a previously studied database. Although pK(a) lowering interactions at the N-termini are similar in nature to those encountered in other protein regions, pK(a) lowering backbone and side-chain hydrogen bonds appear in greater number at the N-termini. For both Asp and Glu, there are about 0.5 more hydrogen bonds per residue at the N-termini than in other protein regions, which can be used to explain their lower than average pK(a) values. Using a QM-based pK(a) prediction model, we investigate the chemical environment of the two lowest Asp and the two lowest Glu pK(a) values at the N-termini so as to quantify the effect of various pK(a) determinants. We show that local interactions suffice to account for the acidity of carboxyl residues at the N-termini. The effect of the helix dipole on carboxyl pK(a) values, if any, is marginal. Backbone amide hydrogen bonds constitute the single biggest contributor to the lowest carboxyl pK(a) values at the N-termini. Their estimated pK(a) lowering effects range from about 1.0 to 1.9 pK(a) units. 相似文献
6.
Timothy Lockwich Lawrence M. Mertz Indu S. Ambudkar 《Molecular and cellular biochemistry》1993,126(2):143-150
Divalent cation permeability of rat parotid gland basolateral plasma membranes was examined in dispersed parotid acini (by Ca2+ or Mn2+ entry) and in isolated basolateral plasma membrane vesicles (BLMV, by45Ca2+ influx). Mn2+ entry (fura2 quenching) was about 1.6 fold higher in internal Ca2+ pool-depleted acini (Ca2+-depl acini) than in unstimulated cells. Mn2+ entry into Ca2+-depl acini was increased at external pH>7.4 and decreased at pH<7.4. Pretreatment of Ca2+-depl acini with the relatively hydrophobic carboxylic group reagent, N,N-dicyclohexylcarbodiimide (DCCD, 50 M for 30 min) resulted in the inhibition of Mn2+ entry into Ca2+-depl acini to unstimulated levels. Another hydrophobic carboxyl group reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and the relatively hydrophilic carboxyl group reagents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMCD) did not affect Mn2+ entry.Similar to the effects in intact acini, Ca2+ influx into BLMV was decreased when the external pH was lowered below 7.4. Also DCCD (5 mM, 30 min), but not EEDQ, decreased (40%) Ca2+ influx in BLMV. However, unlike in acini, the hydrophilic reagents, EDC, EAC, and CMCD decreased Ca2+ permeability in BLMV and the effects were nonadditive with the decrease induced by DCCD. The aggregate effects of carboxyl group reagents on the Ca2+ and Mn2+ permeability in BLMV and intact acini, respectively, suggest that a critical carboxyl group (most likely accessible from the cytoplasmic side of the plasma membrane) is involved in divalent cation flux in rat parotid acinar cells. 相似文献
7.
Chimenti MS Castañeda CA Majumdar A García-Moreno E B 《Journal of molecular biology》2011,405(2):361-377
The side chains of Lys66, Asp66, and Glu66 in staphylococcal nuclease are fully buried and surrounded mainly by hydrophobic matter, except for internal water molecules associated with carboxylic oxygen atoms. These ionizable side chains titrate with pKa values of 5.7, 8.8, and 8.9, respectively. To reproduce these pKa values with continuum electrostatics calculations, we treated the protein with high dielectric constants. We have examined the structural origins of these high apparent dielectric constants by using NMR spectroscopy to characterize the structural response to the ionization of these internal side chains. Substitution of Val66 with Lys66 and Asp66 led to increased conformational fluctuations of the microenvironments surrounding these groups, even under pH conditions where Lys66 and Asp66 are neutral. When Lys66, Asp66, and Glu66 are charged, the proteins remain almost fully folded, but resonances for a few backbone amides adjacent to the internal ionizable residues are broadened. This suggests that the ionization of the internal groups promotes a local increase in dynamics on the intermediate timescale, consistent with either partial unfolding or increased backbone fluctuations of helix 1 near residue 66, or, less likely, with increased fluctuations of the charged side chains at position 66. These experiments confirm that the high apparent dielectric constants reported by internal Lys66, Asp66, and Glu66 reflect localized changes in conformational fluctuations without incurring detectable global structural reorganization. To improve structure-based pKa calculations in proteins, we will need to learn how to treat this coupling between ionization of internal groups and local changes in conformational fluctuations explicitly. 相似文献
8.
Summary Voltage-clamped single nerve fibers of the frogRana esculenta were treated with the carboxyl group activating reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in the presence of different primary amines and without added amine. Carboxyl groups form stable amide bonds with primary amines in the presence of EEDQ. EEDQ treatment reduced the sodium current considerably and irreversibly, regardless of the presence of a primary amine in the Ringer's solution. The potassium current was also reduced. After modification the reduced sodium currents inactivated slowly and incompletely. The descending branch of the sodium current-voltage relation,I
Na(E), was shifted along the voltage axis in the depolarizing direction. The size of the shift was strongly dependent on the amine present during modification with EEDQ. The voltage-dependence of sodium inactivation,h
x
(E), was shifted to more positive values of membrane potential by EEDQ in the presence of ethylenediamine (11 mV) and glucosamine (3 mV). In contrast, a small shift to more negative potentials occurred in the presence of taurine (–3 mV) or without the addition of an amine (–2 mV). A tenfold increase of the calcium concentration still shifted theI
Na(E) andh
x
(E) curves of the chemically modified fibers. However, these shifts were smaller than those observed on untreated fibers. The currents remaining after the modification were completely blocked by tetrodotoxin; no change of the reversal potential occurred. 相似文献
9.
The nature of the denatured state ensemble has been controversial for decades owing, in large part, to the difficulty in characterizing the structure and energetics of denatured state interactions. There is increasing evidence for relatively non-specific hydrophobic clustering in the denatured states of some proteins but other types of interactions are much less well characterized. Here, we report the characterization of highly specific electrostatic interactions in the denatured state of a small alpha-beta protein, the N-terminal domain of the ribosomal protein L9 (NTL9). Mutation of Lys12 to Met has been shown to increase the stability of NTL9 significantly through the disruption of denatured state interactions. Here, we describe the analysis of the pH-dependent stability of 13 mutants designed to probe the nature of the Lys12 denatured state interaction. Lys12 is located in a lysine-rich region of the protein but analysis of a set of Lys to Met mutants shows that it plays a unique role in the denatured state. Analysis of mutants of all of the acidic residues in NTL9 shows that Lys12 forms a specific non-native electrostatic interaction with Asp8 in the denatured state ensemble. Thus the distribution of charge-charge interactions in the denatured state ensemble of NTL9 appears to be biased by few key interactions and is very different from that expected in a random coil. We propose that these interactions are not encoded by local sequence effects but rather reflect interactions among residues more distant in sequence. These results demonstrate that electrostatic as well as hydrophobic interactions can play an important role in the denatured state ensemble. 相似文献
10.
Paper electrophoresis has been used over the pH range 1.2 to 10.4 to measure apparent pK(a) values for malvidin-3-O-glucoside of pK(a(1)) 1.76+/-0.07, pK(a(2)) 5.36+/-0.04, and pK(a(3)) 8.39+/-0.07. Using solvent partitioning between buffered aqueous solutions and n-octanol, several micro-pK(a) constants for malvidin-3-O-glucoside were also identified, highlighting the complex nature of malvidin-3-glucoside equilibria. As a nonspectrophotometric procedure, the charge-dependent electrophoretic mobility method provided independent information on the net charge and color of anthocyanin species at wine pH (ca. 3.6). At this pH, the color of malvidin-3-glucoside in red wines is consistent only with the uncharged quinonoidal base as a major colored component of the equilibria. 相似文献
11.
It is often assumed that the peptide backbone forms a substantial number of additional hydrogen bonds when a protein unfolds. We challenge that assumption in this article. Early surveys of hydrogen bonding in proteins of known structure typically found that most, but not all, backbone polar groups are satisfied, either by intramolecular partners or by water. When the protein is folded, these groups form approximately two hydrogen bonds per peptide unit, one donor or acceptor for each carbonyl oxygen or amide hydrogen, respectively. But when unfolded, the backbone chain is often believed to form three hydrogen bonds per peptide unit, one partner for each oxygen lone pair or amide hydrogen. This assumption is based on the properties of small model compounds, like N-methylacetamide, or simply accepted as self-evident fact. If valid, a chain of N residues would have approximately 2N backbone hydrogen bonds when folded but 3N backbone hydrogen bonds when unfolded, a sufficient difference to overshadow any uncertainties involved in calculating these per-residue averages. Here, we use exhaustive conformational sampling to monitor the number of H-bonds in a statistically adequate population of blocked polyalanyl-six-mers as the solvent quality ranges from good to poor. Solvent quality is represented by a scalar parameter used to Boltzmann-weight the population energy. Recent experimental studies show that a repeating (Gly-Ser) polypeptide undergoes a denaturant-induced expansion accompanied by breaking intramolecular peptide H-bonds. Results from our simulations augment this experimental finding by showing that the number of H-bonds is approximately conserved during such expansion⇋compaction transitions. 相似文献
12.
The role of a conserved tyrosine residue in high-potential iron sulfur proteins. 总被引:1,自引:0,他引:1
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S. G. Iwagami A. L. Creagh C. A. Haynes M. Borsari I. C. Felli M. Piccioli L. D. Eltis 《Protein science : a publication of the Protein Society》1995,4(12):2562-2572
Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 O delta 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-59/Val-58 peptide bond, thereby perturbing Gly-46. The delta delta GDapp of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring. 相似文献
13.
Several small ribozymes employ general acid–base catalysis as a mechanism to enhance site-specific RNA cleavage, even though the functional groups on the ribonucleoside building blocks of RNA have pKa values far removed from physiological pH. The rate of the cleavage reaction is strongly affected by the identity of the metal cation present in the reaction solution; however, the mechanism(s) by which different cations contribute to rate enhancement has not been determined. Using the Neurospora VS ribozyme, we provide evidence that different cations confer particular shifts in the apparent pKa values of the catalytic nucleobases, which in turn determines the fraction of RNA in the protonation state competent for general acid–base catalysis at a given pH, which determines the observed rate of the cleavage reaction. Despite large differences in observed rates of cleavage in different cations, mathematical models of general acid–base catalysis indicate that k1, the intrinsic rate of the bond-breaking step, is essentially constant irrespective of the identity of the cation(s) in the reaction solution. Thus, in contrast to models that invoke unique roles for metal ions in ribozyme chemical mechanisms, we find that most, and possibly all, of the ion-specific rate enhancement in the VS ribozyme can be explained solely by the effect of the ions on nucleobase pKa. The inference that k1 is essentially constant suggests a resolution of the problem of kinetic ambiguity in favor of a model in which the lower pKa is that of the general acid and the higher pKa is that of the general base. 相似文献
14.
Previous studies of ubiquitin disclosed numerous charge-charge interactions on the protein's surface. To investigate how neighboring residues influence the strength of these interactions, double-mutant cycles are combined with pK(a) determinations by 2D NMR. More specifically, the environment around the Asp21-Lys29 ion pair has been altered through mutations at position 25, which is an asparagine in mammalian ubiquitin and a positively-charged residue in many other ubiquitin-like proteins. The pK(a) value of Asp21 decreases by 0.4 to 0.7 pH unit when Asn25 is substituted with a positively charged residue, suggesting a new and favorable ion pair interaction between positions 21 and 25. However, analysis of double mutants reveals that the favorable interaction between Asp21 and Lys29 is weakened when position 25 is a positively charged residue. Interestingly, while the pK(a) value of His25 in the N25H variant agrees with model compound values, additional mutants reveal that this agreement is fortuitous, resulting from a balance of favorable and unfavorable interactions; similar results were observed previously for Glu34 in ubiquitin and His8 in staphylococcal nuclease. Ionizable groups may thus have pK(a) values similar to model compound values and yet still be involved in significant interactions with other protein groups. One surprising result of introducing positively charged residues at position 25 is a new interaction between Lys29 and Glu18, an interaction not present in wild-type ubiquitin. This unanticipated result illustrates a key advantage of using NMR to determine pK(a) values for many residues simultaneously in the variant proteins. Overall, the strength of an interaction between two residues at the surface of ubiquitin is sensitive to the identity of neighboring residues. The results also demonstrate that relatively conservative and common point mutations such as substitutions of polar with charged residues and vice versa can have effects on interactions beyond the site of mutation per se. 相似文献
15.
S. F. Betz 《Protein science : a publication of the Protein Society》1993,2(10):1551-1558
An understanding of the forces that contribute to stability is pivotal in solving the protein-folding problem. Classical theory suggests that disulfide bonds stabilize proteins by reducing the entropy of the denatured state. More recent theories have attempted to expand this idea, suggesting that in addition to configurational entropic effects, enthalpic and native-state effects occur and cannot be neglected. Experimental thermodynamic evidence is examined from two sources: (1) the disruption of naturally occurring disulfides, and (2) the insertion of novel disulfides. The data confirm that enthalpic and native-state effects are often significant. The experimental changes in free energy are compared to those predicted by different theories. The differences between theory and experiment are large near 300 K and do not lend support to any of the current theories regarding the stabilization of proteins by disulfide bonds. This observation is a result of not only deficiencies in the theoretical models but also from difficulties in determining the effects of disulfide bonds on protein stability against the backdrop of numerous subtle stabilizing factors (in both the native and denatured states), which they may also affect. 相似文献
16.
We study the thermodynamic behavior of a model protein with 54 amino acidsthat is designed to form a three-helix bundle in its native state. The model contains three types of amino acids and five to six atoms per amino acid, and has the Ramachandran torsion angles as its only degrees of freedom.The force field is based on hydrogen bonds and effective hydrophobicity forces. We study how the character of the collapse transition depends on the strengths of these forces. For a suitable choice of these two parameters, it is found that the collapse transition is first-order-like and coincides with the folding transition. Also shown is that the corresponding one- and two-helix segments make less stable secondary structure than the three-helix sequence. 相似文献
17.
Non-traditional C-H cdots, three dots, centered Y hydrogen bonds, in which a carbon atom acts as the hydrogen donor and an electronegative atom Y (Y=N, O or S) acts as the acceptor, have been reported in proteins, but their importance in protein structures is not well established. Here, we present the results of three computational tests that examine the significance of C-H cdots, three dots, centered Y bonds involving the C(alpha) in proteins. First, we compared the number of C(alpha)-H cdots, three dots, centered Y bonds in native structures with two sets of compact, energy-minimized decoy structures. The decoy structures contain about as many C(alpha)-H cdots, three dots, centered Y bonds as the native structures, indicating that the constraints of chain connectivity and compactness can lead to incidental formation of C(alpha)-H cdots, three dots, centered Y bonds. Secondly, we examined whether short C(alpha)-H cdots, three dots, centered Y bonds have a tendency to be linear, as is expected for a cohesive hydrogen-bonding interaction. The native structures do show this trend, but so does one of the decoy sets, suggesting that this criterion is also not sufficient to indicate a stabilizing interaction. Finally, we examined the preference for C(alpha)-H cdots, three dots, centered Y bond donors to be near to strong hydrogen bond acceptors. In the native proteins, the alpha protons attract strong acceptors like oxygen atoms more than weak acceptors. In contrast, hydrogen bond donors in the decoy structures do not distinguish between strong and weak acceptors. Thus, any individual C(alpha)-H cdots, three dots, centered Y bond may be fortuitous and occur due to the polypeptide connectivity and compactness. Taken collectively, however, C(alpha)-H cdots, three dots, centered Y bonds provide a weakly cohesive force that stabilizes proteins. 相似文献
18.
Analyzing the stability of a multimeric protein is challenging because of the intrinsic difficulty in handling the mathematical model for the folded multimer-unfolded monomer equilibrium. To circumvent this problem, we introduce the concept of effective stability, DeltaGeff (= -RTlnKeff), where Keff is the equilibrium constant expressed in monomer units. Analysis of the denaturant effect on DeltaGeff gives new insight into the stability of multimeric proteins. When a multimeric protein is mostly folded, the dependence of effective stability on denaturant concentration (effective m-value) is simply the m-value of its monomeric unit. However, when the protein is mostly unfolded, its stability depends on denaturant concentration with the m-value of its multimeric form. We also find that the effective m-value at the Cm is a good approximation of the apparent m-value determined by fitting the equilibrium unfolding data from multimeric proteins with a two-state monomer model. Moreover, when the m-value of a monomeric unit is estimated from its size, the effective stability of a multimeric protein can be determined simply from Cm and this estimated m-value. These simple and intuitive approaches will allow a facile analysis of the stability of multimeric proteins. These analyses are also applicable for high-throughput analysis of protein stability on a proteomic scale. 相似文献
19.
Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB(SS). The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure. pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(a)(U) values in unfolded peptides with partial sequences of AB(SS) were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (-5.6 kJ mol(-1)) and two are interacting with the positive alpha-helix dipoles and are thus stabilizing (+3.8 kJ mol(-1)) in charged form. The two His residues positioned in the C-terminal moiety of AB(SS) interact with the negative alpha-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (-2.6 kJ mol(-1)). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (-1.1 kJ mol(-1)). The sum of the individual contributions of protonated Glu and His to the higher stability of AB(SS) at acidic pH (-5.4 kJ mol(-1)) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (-5.3 kJ mol(-1)). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys-->norleucine, Arg-->norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the pK(a) of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge-charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability. 相似文献
20.
Barrera FN Weerakkody D Anderson M Andreev OA Reshetnyak YK Engelman DM 《Journal of molecular biology》2011,413(2):359-10222
We have used pHLIP® [pH (low) insertion peptide] to study the roles of carboxyl groups in transmembrane (TM) peptide insertion. pHLIP binds to the surface of a lipid bilayer as a disordered peptide at neutral pH; when the pH is lowered, it inserts across the membrane to form a TM helix. Peptide insertion is reversed when the pH is raised above the characteristic pKa (6.0). A key event that facilitates membrane insertion is the protonation of aspartic acid (Asp) and/or glutamic acid (Glu) residues, since their negatively charged side chains hinder membrane insertion at neutral pH. In order to gain mechanistic understanding, we studied the membrane insertion and exit of a series of pHLIP variants where the four Asp residues were sequentially mutated to nonacidic residues, including histidine (His). Our results show that the presence of His residues does not prevent the pH-dependent peptide membrane insertion at ∼ pH 4 driven by the protonation of carboxyl groups at the inserting end of the peptide. A further pH drop leads to the protonation of His residues in the TM part of the peptide, which induces peptide exit from the bilayer. We also find that the number of ionizable residues that undergo a change in protonation during membrane insertion correlates with the pH-dependent insertion into the lipid bilayer and exit from the lipid bilayer, and that cooperativity increases with their number. We expect that our understanding will be used to improve the targeting of acidic diseased tissue by pHLIP. 相似文献