Nonpolar Amino Acid Residues Research Articles

The essential roles of protein–protein interaction in sigma-1 receptor functions

The essential roles of protein–protein interaction in sigma-1 receptor functions

Individual degrees of freedom and the solvation properties of water

Using molecular dynamics simulations in conjunction with home-developed Split Integration Symplectic Method we effectively decouple individual degrees of freedom of water molecules and connect them to corresponding thermostats. In this way, we facilitate elucidation of structural, dynamical, spectral, and hydration properties of bulk water at any given combination of rotational, translational, and vibrational temperatures. Elevated rotational temperature of the water medium is found to severely hinder hydration of polar molecules, to affect hydration of ionic species in a nonmonotonous way and to somewhat improve hydration of nonpolar species. As proteins consist of charged, polar, and nonpolar amino-acid residues, the developed methodology is also applied to critically evaluate the hypothesis that the overall decrease in protein hydration and the change in the subtle balance between hydration of various types of amino-acid residues provide a plausible physical mechanism through which microwaves enhance aberrant protein folding and aggregation.

Design of Potent, Non‐Toxic Antimicrobial Agents Based upon the Structure of the Frog Skin Peptide, Temporin‐1CEb from Chinese Brown Frog, Rana chensinensis

Temporin-1CEb shows antimicrobial activity against Gram-positive bacteria, but its therapeutic potential is limited by its haemolysis. In this study, eight temporin-1CEb analogues with altered cationicities and hydrophobicities were synthesized. Increasing cationicity and amphipathicity by substituting neutral and non-polar amino acid residues on the hydrophilic face of the α-helix by five or six lysines increased antimicrobial potency approximately 10-fold to 40-fold, although when the number of positive charges was increased from +6 to +7, the antimicrobial potency was not additionally enhanced. The substitution of an l-lysine with a d-lysine, meanwhile maintaining the net charge and the mean hydrophobicity values, had only a minor effect on its antimicrobial activity, whereas significantly led a decrease in its haemolytic activity. Of all the peptides, l-K6 has the best potential as an antimicrobial agent because its antimicrobial activity against both Gram-positive and Gram-negative bacteria is substantial, and its haemolytic activity is negligible. l-K6 adopts an α-helix in 50% trifluoroethanol/water and 30 mm SDS solutions. l-K6 killed 99.9% of E.coli and S.aureus at 4× MIC in 60 min, and its postantibiotic effect was >5 h. l-K6 affects the integrity of E.coli and S.aureus plasma membranes by rapidly inducing membrane depolarization.

Non-canonical interactions of porphyrins in porphyrin-containing proteins

In this study we have described the noncanonical interactions between the porphyrin ring and the protein part of porphyrin-containing proteins to better understand their stabilizing role. The analysis reported in this study shows that the predominant type of non-canonical interactions at porphyrins are CH····O and CH····N interactions, with a small percentage of CH···π and noncanonical interactions involving sulfur atoms. The majority of non-canonical interactions are formed from side-chains of charged and polar amino acids, whereas backbone groups are not frequently involved. The main-chain noncanonical interactions might be slightly more linear than the side-chain interactions, and they have somewhat shorter median distances. The analysis, performed in this study, shows that about 44% of the total interactions in the dataset are involved in the formation of multiple (furcated) noncanonical interactions. The high number of porphyrin-water interactions show importance of the inclusion of solvent in protein-ligand interaction studies. Furthermore, in the present study we have observed that stabilization centers are composed predominantly from nonpolar amino acid residues. Amino acids deployed in the environment of porphyrin rings are deposited in helices and coils. The results from this study might be used for structure-based porphyrin protein prediction and as scaffolds for future porphyrin-containing protein design.

Exploring molecular docking and QSAR studies of plasmepsin-II inhibitor di-tertiary amines as potential antimalarial compounds

We report molecular docking and quantitative structure–activity relationship (QSAR) studies on plasmepsin-II inhibitor di-tertiary amines as antimalarial agents. The docking studies suggest that the molecules bind with a pocket of the enzyme formed by some non-polar amino acid residues (Ala219, Gly36, Gly216, Phe111, Phe120, Val78, Ileu32, Ileu123, Ileu290, Ileu300, Leu131 and Leu292) and some polar amino acid residues (Asp34, Asp214, Ser37, Ser218, Thr217, Tyr77 and Tyr192). Different QSAR models, obtained using various three-dimensional, physicochemical and/or topological descriptors and showing cross-validated predictive variance values (Q 2) ranging from 0.487 to 0.664 and the external predictive variance values () for the test set ranging from 0.534 to 0.579, support the statistical validity of the QSAR models.

Identification of novel and recurrent mutations in Tunisian haemophilia B patients

Haemophilia B disease is a recessively inherited X-linked bleeding disorder which results from deficiency of factor IX (F9). Haemophilia B has a frequency of approximately 1 in 25 000 men worldwide [1]. Haemophilia B results from heterogeneous mutations spread throughout the F9 gene [2]. According to the World Federation of Hemophilia Report on the annual global survey 2007, 51 haemophilia B in Tunisia have been reported [3]. In this first study on Tunisian haemophilia B, we report the molecular analysis of 16 unrelated haemophilia B families. Patients involved in this study were from the Hemophilia Treatment Center, Aziza Othmana hospital, Tunisia. Informed consent was obtained from all patients. Molecular analysis was performed using the following strategy: polymerase chain reactions for the entire coding sequence of the F9 gene were prepared as described previously [4]. The mutation detection protocol was performed by dHPLC on a WAVE DNA Fragment Analysis System (Transgenomics, San Jose, USA). Altered profiles detected by dHPLC were sequenced using ABI Dye Terminator Cycle Sequencing (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) and analysed using a capillary sequencer Genetic Analyser ABI PRISM310 (Perkin-Elmer Applied Biosystems, Foster City, CA, USA [4]. Results were analysed using BLAST (http://www.ncbi.nlm. nih.gov/blast) program against the normal F9 gene sequence (GenBank Accession No. K02402) and the mutations were compared with the haemophilia B mutation database (http://www.umds.ac.uk/molgen). To evaluate the nature of missense mutations, we used PolyPhen (Polymorphism Phenotyping) (http://genetics.bwh.harvard.edu/pph). Our cohort is composed of 30 patients belonging to 16 unrelated families who represent 60% of total haemophilia B Tunisian population. A total of 15 different mutations were detected (Table 1), except for one family that did not show any mutations. In addition, the polymorphism g.20421A>G in exon 6 was also identified in five families. Five novel mutations were identified in five patients including 2 missense mutations, 1 nonsense mutation, 1 splice site mutation and one small deletion. For patient Hb 2, a deletion of CAG sequence from the 17795 to 17797 position inducing the loss of the last acid Ala173 in exon 5 (The numbering of the amino acids is according to the Swiss-Prot PZES (P00740)). For patient Hb10, a T to A substitution at nucleotide position 113 which changes a Cys acid in a codon stop (Cys27X) in exon 1, which will result in nonsense-mediated RNA decay and produce a severe phenotype as no protein will be translated, has been revealed. Patient Hb12 shows an acceptor splice substitution at the position 10507 in intron 4 (+2T > C). For Patient Hb14, a substitution of T to A at the position 31286 in exon 8, change the Cys 435 to Ser. Patient Hb16 present a substitution of G to A at the position 30932 in exon 8, which change Ala 317 to Thr. Replacement of a non-polar amino acid residue by a polar one is likely to affect the function, secretion or stability of the protein. Using PolyPhen these two mutations are predicted to be probably damaging with a score of 1.000 and 0.995 respectively. The question of whether these two candidate mutations Cys435Ser and Ala317Thr are pathogenic and alter the three-dimensional structure and function of F9 protein needs further investigation. However, as the latter mutations along with, Ala173Del, Ala317Thr and Cys435Ser occurred at amino acid residues highly conserved among different species, they may be involved in the F9 destabilization. Compared with previously published reports [5], we found that the two deletions identified in our patients bearing a severe disease. However, in our patient cohort the two nonsense mutations were associated with different phenotypes, severe and moderate disease respectively in patient Hb17(FIX:C level of 4) which is at variance with the majority of entries on the haemophilia B database for this mutation (which cite FIX:C and antigen levels of <1 for most of the 56 examples). Our observation in Hb17 is actually the exception for this particular mutation and for most nonsense mutations in general. In patient Hb3, we could not detect any mutations in the F9 gene using first dHPLC (no altered profile was observed) then sequencing. It is possible that this family might have pathological translocation, duplication or inversion in the factor IX gene leading to the disease. Further investigation is needed. To our knowledge this study is the first comprehensive molecular analysis of haemophilia B patients in Tunisia. Five novel mutations were identified and our data are globally in agreement with other reports in the international database. When requested, the data obtained from this study will be used for carrier testing and prenatal diagnosis. The identification of the mutations can also be used to estimate the risk of inhibitor development. It can also be valuable when planning future studies including gene therapy.

Conformational preferences of non-polar amino acid residues: An additional factor in amyloid formation

Amyloid consists of β-sheet polymers and is associated with disease and with functional assemblies. Amyloid-forming proteins differ widely in native structures and sequences. We describe here how conformational preferences of non-polar amino acid residues can affect amyloid formation. The most non-polar residues promote either β-strands (Val, Ile, Phe, and Cys, VIFC) or α-helices (Leu, Ala, and Met, LAM), while the most polar residues promote only α-helices. For 12 proteins associated with disease, the localizations of the amyloid core regions are known. Eleven of these contain segments that are biased for VIFC, but essentially lack segments that are biased for LAM. For the amyloid β-peptide associated with Alzheimer’s disease and an amyloidogenic fragment of the prion protein, observed effects of mutations support that VIFC bias favors formation of β-sheet aggregates and amyloid, while LAM bias prevents it. VIFC and LAM profiles combine information on secondary structure propensities and polarity, and add a simple criterion to the prediction of amyloidogenic regions.

Chemometric modeling, docking and in silico design of triazolopyrimidine-based dihydroorotate dehydrogenase inhibitors as antimalarials

In the present work, QSAR and molecular docking studies have been performed on triazolopyrimidine-based dihydroorotate dehydrogenase (DHODH) inhibitors as antimalarial agents. The QSAR studies have been carried out using classical QSAR (physicochemical) approach using linear free energy related (LFER) model and molecular shape analysis using shape, spatial, electronic, thermodynamic and structural descriptors. Docking studies suggest that the 2-methyltriazolopyrimidine ring interacts with some polar and some nonpolar amino acids whereas the substituted phenyl ring binds with a hydrophobic pocket of the enzyme formed by some nonpolar amino acid residues. According to QSAR and molecular docking studies, we have designed some new compounds which showed good in silico predicted activity as per the developed QSAR models.

Estimation of global structural and transport properties of peptides through the modeling of their CZE mobility data

Peptide electrophoretic mobility data are interpreted through a physicochemical CZE model, providing estimates of the equivalent hydrodynamic radius, hydration, effective and total charge numbers, actual ionizing pK, pH-near molecule and electrical permittivity of peptide domain, among other basic properties. In this study, they are used to estimate some peptide global structural properties proposed, providing thus a distinction among different peptides. Therefore, the solvent drag on the peptide is obtained through a characteristic friction power coefficient of the number of amino acid residues, defined from the global chain conformation in solution. As modeling of the effective electrophoretic mobility of peptides is carried out in terms of particle hydrodynamic size and shape coupled to hydration and effective charge, a packing dimension related to chain conformation within the peptide domain may be defined. In addition, the effective and total charge number fractions of peptides provide some clues on the interpretation of chain conformations within the framework of scaling laws. Furthermore, the model estimates transport properties, such as sedimentation, friction and diffusion coefficients. As the relative numbers of ionizing, polar and non-polar amino acid residues vary in peptides, their global structural properties defined here change appreciably. Needs for further research are also discussed.

Terpestacin Inhibits Tumor Angiogenesis by Targeting UQCRB of Mitochondrial Complex III and Suppressing Hypoxia-induced Reactive Oxygen Species Production and Cellular Oxygen Sensing

Cellular oxygen sensing is required for hypoxia-inducible factor-1alpha stabilization, which is important for tumor cell survival, proliferation, and angiogenesis. Here we find that terpestacin, a small molecule previously identified in a screen of microbial extracts, binds to the 13.4-kDa subunit (UQCRB) of mitochondrial Complex III, resulting in inhibition of hypoxia-induced reactive oxygen species generation. Consequently, such inhibition blocks hypoxia-inducible factor activation and tumor angiogenesis in vivo, without inhibiting mitochondrial respiration. Overexpression of UQCRB or its suppression using RNA interference demonstrates that it plays a crucial role in the oxygen sensing mechanism that regulates responses to hypoxia. These findings provide a novel molecular basis of terpestacin targeting UQCRB of Complex III in selective suppression of tumor progression.

Structural Determination and Tryptophan Fluorescence of Heterokaryon Incompatibility C2 Protein (HET-C2), a Fungal Glycolipid Transfer Protein (GLTP), Provide Novel Insights into Glycolipid Specificity and Membrane Interaction by the GLTP Fold

HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated. X-ray diffraction (1.9 A) revealed a GLTP fold with all key sugar headgroup recognition residues (Asp(66), Asn(70), Lys(73), Trp(109), and His(147)) conserved and properly oriented for glycolipid binding. Far-UV CD showed secondary structure dominated by alpha-helices and a cooperative thermal unfolding transition of 49 degrees C, features consistent with a GLTP fold. Environmentally induced optical activity of Trp/Tyr/Phe (2:4:12) detected by near-UV CD was unaffected by membranes containing glycolipid but was slightly altered by membranes lacking glycolipid. Trp fluorescence was maximal at approximately 355 nm and accessible to aqueous quenchers, indicating free exposure to the aqueous milieu and consistent with surface localization of the two Trps. Interaction with membranes lacking glycolipid triggered significant decreases in Trp emission intensity but lesser than decreases induced by membranes containing glycolipid. Binding of glycolipid (confirmed by electrospray injection mass spectrometry) resulted in a blue-shifted emission wavelength maximum (approximately 6 nm) permitting determination of binding affinities. The unique positioning of Trp(208) at the HET-C2 C terminus revealed membrane-induced conformational changes that precede glycolipid uptake, whereas key differences in residues of the sugar headgroup recognition center accounted for altered glycolipid specificity and suggested evolutionary adaptation for the simpler glycosphingolipid compositions of filamentous fungi.

Docking and 3D-QSAR studies of diverse classes of human aromatase (CYP19) inhibitors

Aromatase (cytochrome 19) inhibitors have emerged as promising candidates for treatment of breast cancer. In search of potent aromatase inhibitors, docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies using molecular shape, spatial, electronic, structural and thermodynamic descriptors have been performed on a diverse set of compounds having human aromatase inhibitory activities. An attempt has also been made to include two-dimensional (2D) descriptors in the QSAR studies. The chemometric tools used for model development are genetic function approximation (GFA) and genetic partial least squares (G/PLS). The docking study shows that the important interacting amino acids in the active site cavity are Met374, Arg115, Ile133, Ala306, Thr310, Asp309, Val370 and Ser478. One or more hydrogen bond formation with Met374 is one of the essential requirements for the ligands for optimum aromatase inhibition. The binding is further stabilized by van der Waals interactions with a few non-polar amino acid residues in the active site. The developed QSAR models indicate the importance of different shape, Jurs parameters, structural parameters, topological branching index and E-state index for different fragments. The results obtained from the QSAR analysis are supported by our docking observations. There should be one or two hydrogen bond acceptor groups (like -NO2, -CN) and optimal hydrophobicity for ideal aromatase inhibitors. A GFA model with spline option obtained using 3D descriptors was found to be the best model based on internal validation (Q2=0.668) while the best (externally) predictive model was a GFA model with spline option using combined set (2D and 3D) descriptors (Rpred2=0.687). Based on rm2(overall) criterion, the best model was a G/PLS model (using 3D descriptors) with spline option (rm2(overall)=0.606).

Ligand Binding and Hydration in Protein Misfolding: Insights from Studies of Prion and p53 Tumor Suppressor Proteins

Protein misfolding has been implicated in a large number of diseases termed protein- folding disorders (PFDs), which include Alzheimer’s disease, Parkinson’s disease, transmissible spongiform encephalopathies, familial amyloid polyneuropathy, Huntington’s disease, and type II diabetes. In these diseases, large quantities of incorrectly folded proteins undergo aggregation, destroying brain cells and other tissues.The interplay between ligand binding and hydration is an important component of the formation of misfolded protein species. Hydration drives various biological processes, including protein folding, ligand binding, macromolecular assembly, enzyme kinetics, and signal transduction. The changes in hydration and packing, both when proteins fold correctly or when folding goes wrong, leading to PFDs, are examined through several biochemical, biophysical, and structural approaches. Although in many cases the binding of a ligand such as a nucleic acid helps to prevent misfolding and aggregation, there are several examples in which ligands induce misfolding and assembly into amyloids. This occurs simply because the formation of structured aggregates (such as protofibrillar and fibrillar amyloids) involves decreases in hydration, formation of a hydrogen-bond network in the secondary structure, and burying of nonpolar amino acid residues, processes that also occur in the normal folding landscape. In this Account, we describe the present knowledge of the folding and misfolding of different proteins, with a detailed emphasis on mammalian prion protein (PrP) and tumoral suppressor protein p53; we also explore how ligand binding and hydration together influence the fate of the proteins.Anfinsen’s paradigm that the structure of a protein is determined by its amino acid sequence is to some extent contradicted by the observation that there are two isoforms of the prion protein with the same sequence: the cellular and the misfolded isoform. The cellular isoform of PrP has a disordered N-terminal domain and a highly flexible, not-well-packed C-terminal domain, which might account for its significant hydration. When PrP binds to biological molecules, such as glycosaminoglycans and nucleic acids, the disordered segments appear to fold and become less hydrated. Formation of the PrP−nucleic acid complex seems to accelerate the conversion of the cellular form of the protein into the disease-causing isoform. For p53, binding to some ligands, including nucleic acids, would prevent misfolding of the protein. Recently, several groups have begun to analyze the folding−misfolding of the individual domains of p53, but several questions remain unanswered. We discuss the implications of these findings for understanding the productive and incorrect folding pathways of these proteins in normal physiological states and in human disease, such as prion disorders and cancer. These studies are shown to lay the groundwork for the development of new drugs.

Docking and 3D QSAR studies of protoporphyrinogen oxidase inhibitor 3H-pyrazolo[3,4-d][1,2,3]triazin-4-one derivatives

Docking and three dimensional quantitative structure - activity relationship (3D-QSAR) studies have been performed for protoporphyrinogen oxidase (PPO) inhibitor 3H-pyrazolo[3,4-d][1,2,3]triazin-4-one analogues which are potential herbicides to protect agricultural products from unwanted weeds. The 3D-QSAR studies have been carried out using shape, spatial, electronic and molecular field descriptors along with a few structural parameters. The chemometric tools used for the analyses are genetic function approximation (GFA), partial least squares (PLS) and genetic partial least squares (G/PLS). The whole data set (n = 34) was divided into a training set (75% of the data set) and a test set (remaining 25%) on the basis of K-means clustering technique applied on topological, spatial and electronic descriptor matrix. Models developed from the training set were used to predict the activity of the test set compounds. All the models have been validated internally, externally and by Y-randomization technique. Docking studies suggest that the molecules bind with a hydrophobic pocket of the enzyme formed by some nonpolar amino acid (Ile168, Ile311, Ile412, Met365, Phe65 and Val164) residues. The QSAR studies suggest that for better activity the molecules should have symmetrical shape in the 3D space. For better PPO inhibitory activity, there should be a balance between the electrophilic and nucleophilic characters of the inhibitors. The charged surface area descriptors suggest that, the positive charge distributed over a large surface area may enhance the activity. Molecular field probes reflect that increase in steric volume and positively charged surface area may enhance the herbicidal activity.

Characterization of biomimetc material, Mussel adhesive protein, using computer tools and servers.

In this paper six different mussel adhesion proteins (MAPs) retrieved from SwissProt database are analyzed using computer tools and servers. Primary analysis showed that all the MAPs are hydrophobic in nature due to the high content of non-polar amino acid residues. The sequence analysis showed the absence of sulphide bridges and non polar amino acid tryptophan in MAPs. Signal peptide is a common feature in MAPs. The aliphatic index computed by Expasy's Protparm infers that most of the MAPs are stable at a wide range of temperature. Secondary structure analysis showed that MAPs could results in a better interaction with water. The secondary structure analysis showed MAPs contain more βhelices and turns. The SOSUI server predicts one transmembarne region in Mytilus galprovincialis. The predicted transmembrane region was visualized and analyzed using helical wheel plots generated by EMBOSS PepWheel tool. The absence of disulphide bonds in MAPS was confirmed by SYC_REC tools and from 3 Dimensional structures created by Rasmol tool. The cystein position identified by Rasmol tool might be correct as the evaluated parameters (Rampage, CE and ProQ) are within the accepted limits for the model 3D structure. The signal peptide identified by SignalP server showed that they are secretary pathway in localization except in Mytilus edulus, where it is cytoplasmic. Based on the signal peptides reliability class the MAPs are classified into three groups. The phosphorylation sites of serine, threonoine and tyrosine are predicted by NetPhos server. Interestingly tyrosine is the most phosphorylated amino acid in the MAPs; hence it is inferred that MAPs can be purified by using antibodies.

Hydration, charge, size, and shape characteristics of peptides from their CZE analyses

A CZE model is presented for peptide characterization on the basis of well-established physicochemical equations. The effective mobility is used as basic data in the model to estimate relevant peptide properties such as, for instance, hydration, net and total electrical charge numbers, hydrodynamic size and shape, particle average orientation, and pH-microenvironment from the charge regulation phenomenon. Therefore 102 experimental effective mobilities of different peptides are studied and discussed in relation to previous work. An equation for the estimation of peptide hydration as a function of ionizing, polar, and non-polar amino acid residues is included in the model. It is also shown that the shape-orientation factor of peptides may be either lower or higher than one, and its value depends on a complex interplay among total charge number, molar mass, hydration, and amino acid sequence.

Behavior of Acrylodan-Labeled Human Serum Albumin Dissolved in Ionic Liquids

Researchers have reported enzymatic reactions performed in ionic liquids (ILs). Currently very little is known about the behavior of biomolecules in ILs in comparison to their better understood aqueous behavior. We report the temperature-dependent behavior of human serum albumin (HSA) that is site-selectively labeled at cysteine-34 (located in loop 1 of domain I) with a single fluorescent reporter molecule (acrylodan, Ac) when it is dissolved in phosphate buffered saline (PBS) or one of three ILs. In PBS, the Ac reporter motion is always coupled to the global HSA protein motion. In the ILs, loop 1 of domain I appears to be almost completely decoupled from the HSA global motion. As temperature increases, neighboring nonpolar amino acid residues and/or IL components apparently become strongly associated with the Ac rotating body. These results show that protein structure and dynamics in an IL can markedly deviate from that which exists in aqueous media.

Creation of Rhizopus oryzae lipase having a unique oxyanion hole by combinatorial mutagenesis in the lid domain

Combinatorial libraries of the lid domain of Rhizopus oryzae lipase (ROL; Phe88Xaa, Ala91Xaa, Ile92Xaa) were displayed on the yeast cell surface using yeast cell-surface engineering. Among the 40,000 transformants in which ROL mutants were displayed on the yeast cell surface, ten clones showed clear halos on soybean oil-containing plates. Among these, some clones exhibited high activities toward fatty acid esters of fluorescein and contained non-polar amino acid residues in the mutated positions. Computer modeling of the mutants revealed that hydrophobic interactions between the substrates and amino acid residues in the open form of the lid might be critical for ROL activity. Based on these results, Thr93 and Asp94 were further combinatorially mutated. Among 6,000 transformants, the Thr93Thr, Asp94Ser and Thr93Ser, Asp94Ser transformants exhibited a significant shift in substrate specificity toward a short-chain substrate. Computer modeling of these mutants suggested that a unique oxyanion hole, which is composed of Thr85 Ogamma and Ser94 Ogamma, was formed and thus the substrate specificity was changed. Therefore, coupling combinatorial mutagenesis with the cell surface display of ROL could lead to the production of a unique ROL mutant.

Hydration properties and potentials of mean force of nonpolar amino acid residues in water: A pertubation theoretic approach

We use a perturbation approach to calculate the solute-solvent and solute-solute pair correlations for infinitely dilute solutions of nonpolar amino acid side chains in water. In particular, from the solute-solute correlations we derive potentials of mean force for all the distances between different residues of amino acids. Comparison with molecular dynamics simulations performed using GROMOS package shows that the goodness of the approximation varies with the amino acid considered. We discuss in terms of hydrophobicity the different effect that the inclusion of the attractions causes on the solute-water and solute-solute correlations.

Identification of Conserved Prolyl Residue Important for Transport Activity and the Substrate Specificity Range of Yeast Plasma Membrane Na+/H+ Antiporters

Yeast plasma membrane Na+/H+ antiporters are divided according to their substrate specificity in two distinct subfamilies. To identify amino acid residues responsible for substrate specificity determination (recognition of K+), the Zygosaccharomyces rouxii Sod2-22 antiporter (non-transporting K+) was mutagenized and a collection of ZrSod2-22 mutants that improved the KCl tolerance of a salt-sensitive Saccharomyces cerevisiae strain was isolated. Several independent ZrSod2-22 mutated alleles contained the replacement of a highly conserved proline 145 with a residue containing a hydroxyl group (Ser, Thr). Site-directed mutagenesis of Pro145 proved that an amino acid with a hydroxyl group at this position is enough to enable ZrSod2-22p to transport K+. Simultaneously, the P145(S/T) mutation decreased the antiporter transport activity for both Na+ and Li+. Replacement of Pro145 with glycine resulted in a ZrSod2-22p with extremely low activity only for Na+, and the exchange of a charged residue (Asp, Lys) for Pro145 completely stopped the activity. Mutagenesis of the corresponding proline in the S. cerevisiae Nha1 antiporter (Pro146) confirmed that this proline of the fifth transmembrane domain is a critical residue for antiporter function. This is the first evidence that a non-polar amino acid residue is important for the substrate specificity and activity of yeast Nha antiporters.