Effects of Hydrophobic Amino Acid Substitutions on Antimicrobial Peptide Behavior

Antimicrobial peptides (AMPs) are naturally occurring components of the immune system that act against bacteria in a variety of organisms throughout the evolutionary hierarchy. There have been many studies focused on the activity of AMPs using biophysical and microbiological techniques; however, a clear and predictive mechanism toward determining if a peptide will exhibit antimicrobial activity is still elusive, in addition to the fact that the mechanism of action of AMPs has been shown to vary between peptides, targets, and experimental conditions. Nonetheless, the majority of AMPs contain hydrophobic amino acids to facilitate partitioning into bacterial membranes and a net cationic charge to promote selective binding to the anionic surfaces of bacteria over the zwitterionic host cell surfaces. This study explores the role of hydrophobic amino acids using the peptide C18G as a model system. These changes were evaluated for the effects on antimicrobial activity, peptide-lipid interactions using Trp fluorescence spectroscopy, peptide secondary structure formation, and bacterial membrane permeabilization. The results show that while secondary structure formation was not significantly impacted by the substitutions, antibacterial activity and binding to model lipid membranes were well correlated. The variants containing Leu or Phe as the sole hydrophobic groups bound bilayers with highest affinity and were most effective at inhibiting bacterial growth. Peptides with Ile exhibited intermediate behavior while those with Val or α-aminoisobutyric acid (Aib) showed poor binding and activity. The Leu, Phe, and Ile peptides demonstrated a clear preference for anionic bilayers, exhibiting significant emission spectrum shifts upon binding. Similarly, the Leu, Phe, and Ile peptides demonstrated greater ability to disrupt lipid vesicles and bacterial membranes. In total, the data indicate that hydrophobic moieties in the AMP sequence play a significant role in the binding and ability of the peptide to exhibit antibacterial activity.     

Self‐assembly of short peptides composed of only aliphatic amino acids and a combination of aromatic and aliphatic amino acids

The morphology of structures formed by the self‐assembly of short N‐terminal t‐butyloxycarbonyl (Boc) and C‐terminal methyl ester (OMe) protected and Boc‐deprotected hydrophobic peptide esters was investigated. We have observed that Boc‐protected peptide esters composed of either only aliphatic hydrophobic amino acids or aliphatic hydrophobic amino acids in combination with aromatic amino acids, formed highly organized structures, when dried from methanol solutions. Transmission and scanning electron microscopic images of the peptides Boc‐Ile‐Ile‐OMe, Boc‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe and Boc‐Trp‐Ile‐Ile‐OMe showed nanotubular structures. Removal of the Boc group resulted in disruption of the ability to form tubular structures though spherical aggregates were formed. Both Boc‐Leu‐Ile‐Ile‐OMe and H‐Leu‐Ile‐Ile‐OMe formed only spherical nanostructures. Dynamic light scattering studies showed that aggregates of varying dimensions were present in solution suggesting that self‐assembly into ordered structures is facilitated by aggregation in solution. Fourier transform infrared spectroscopy and circular dichroism spectroscopy data show that although all four of the protected peptides adopt well‐defined tertiary structures, upon removal of the Boc group, only H‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe had the ability to adopt β‐structure. Our results indicate that hydrophobic interaction is a very important determinant for self‐assembly and presence of charged and aromatic amino acids in a peptide is not necessary for self‐assembly. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Nonpolar interactions between trans-membrane helical EGF peptide and phosphatidylcholines, sphingomyelins and cholesterol. Molecular dynamics simulation studies.

A molecular dynamics simulation study of four lipid bilayers with inserted trans-membrane helical fragment of epithelial growth factor (EGF) receptor (EGF peptide) was performed. The lipid bilayers differ in their lipid composition and consist of (i) unsaturated phosphatidylcholine (palmitoyloleoylphosphatidylcholine, POPC), (ii) POPC and 20 mol% of cholesterol (Chol), (iii) sphingomyelin (SM) and 20 mol% of Chol, and (iv) SM and 50 mol% of Chol. Only 1 out of 26 residues in the EGF-peptide sequence is polar (Thr). The hydrophobic thickness of each bilayer is different but shorter than the length of the peptide and so, due to hydrophobic mismatch, the inserted peptide is tilted in each bilayer. Additionally, in the POPC bilayer, which is the thinnest, the peptide loses its helical structure in a short three-amino acid fragment. This facilitates bending of the peptide and burying all hydrophobic amino acids inside the membrane core (Figure 1(b)). Bilayer lipid composition affects interactions between the peptide and lipids in the membrane core. Chol increases packing of atoms relative to the peptide side chains, and thus increases van der Waals interactions. On average, the packing around the peptide is higher in SM-based bilayers than POPC-based bilayers but for certain amino acids, packing depends on their position relative to the bilayer center. In the bilayer center, packing is higher in POPC-based bilayers, while in regions closer to the interface packing is higher in SM-based bilayers. In general, amino acids with larger side chains interact strongly with lipids, and thus the peptide sequence is important for the pattern of interactions at different membrane depths. This pattern closely resembles the shape of recently published lateral pressure profiles [Ollila et alJ. Struct. Biol. DOI:10.1016/j.jsb.2007.01.012].     

A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides

Presecretory signal peptides of 39 proteins from diverse prokaryotic and eukaryotic sources have been compared. Although varying in length and amino acid composition, the labile peptides share a hydrophobic core of approximately 12 amino acids. A positively charged residue (Lys or Arg) usually precedes the hydrophobic core. Core termination is defined by the occurrence of a charged residue, a sequence of residues which may induce a beta-turn in a polypeptide, or an interruption in potential alpha-helix or beta-extended strand structure. The hydrophobic cores contain, by weight average, 37% Leu: 15% Ala: 10% Val: 10% Phe: 7% Ile plus 21% other hydrophobic amino acids arranged in a non-random sequence. Following the hydrophobic cores (aligned by their last residue) a highly non-random and localized distribution of Ala is apparent within the initial eight positions following the core: (formula; see text) Coincident with this observation, Ala-X-Ala is the most frequent sequence preceding signal peptidase cleavage. We propose the existence of a signal peptidase recognition sequence A-X-B with the preferred cleavage site located after the sixth amino acid following the core sequence. Twenty-two of the above 27 underlined Ala residues would participate as A or B in peptidase cleavage. Position A includes the larger aliphatic amino acids, Leu, Val and Ile, as well as the residues already found at B (principally Ala, Gly and Ser). Since a preferred cleavage site can be discerned from carboxyl and not amino terminal alignment of the hydrophobic cores it is proposed that the carboxyl ends are oriented inward toward the lumen of the endoplasmic reticulum where cleavage is thought to occur. This orientation coupled with the predicted beta-turn typically found between the core and the cleavage site implies reverse hairpin insertion of the signal sequence. The structural features which we describe should help identify signal peptides and cleavage sites in presumptive amino acid sequences derived from DNA sequences.     

Identification of amino acids involved in protein structural uniqueness: implication for de novo protein design

Structural uniqueness is characteristic of native proteins and is essential to express their biological functions. The major factors that bring about the uniqueness are specific interactions between hydrophobic residues and their unique packing in the protein core. To find the origin of the uniqueness in their amino acid sequences, we analyzed the distribution of the side chain rotational isomers (rotamers) of hydrophobic amino acids in protein tertiary structures and derived deltaS(contact), the conformational-entropy changes of side chains by residue-residue contacts in each secondary structure. The deltaS(contact) values indicate distinct tendencies of the residue pairs to restrict side chain conformation by inter-residue contacts. Of the hydrophobic residues in alpha-helices, aliphatic residues (Leu, Val, Ile) strongly restrict the side chain conformations of each other. In beta-sheets, Met is most strongly restricted by contact with Ile, whereas Leu, Val and Ile are less affected by other residues in contact than those in alpha-helices. In designed and native protein variants, deltaS(contact) was found to correlate with the folding-unfolding cooperativity. Thus, it can be used as a specificity parameter for designing artificial proteins with a unique structure.     

The nature of the hydrophobic binding of small peptides at the bilayer interface: implications for the insertion of transbilayer helices

One method of obtaining useful information about the physical chemistry of peptide/bilayer interactions is to relate thermodynamic parameters of the interactions to structural parameters obtained by diffraction methods. We report here the results of the application of this approach to interactions of hydrophobic tripeptides of the form Ala-X-Ala-O-tert-butyl with lipid bilayers. The thermodynamic constants (delta Gt, delta Ht, and delta St) for the transfer of the tripeptides from water into DMPC vesicles were determined for X = Leu, Phe, and Trp and found to be consistent with those expected for hydrophobic interactions above the phase transition of DMPC. Combining these results with the earlier ones of Jacobs and White [(1986) Biochemistry 25, 2605-2612], the favorable free energies of transfer with different amino acids in the -X- position increase in the order Gly less than Ala less than Leu less than Phe less than Trp in agreement with the Nozaki and Tanford [(1971) J. Biol. Chem. 246, 2211-2217] hydrophobicity scale. Determination of the location of Ala-[2H5]Trp-Ala-O-tert-butyl in oriented DOPC bilayers by neutron diffraction shows that the most hydrophobic peptide of the series is confined to the bilayer headgroup/water region. Refinement of the diffraction measurements shows that only 13% of the tryptophan is associated with the hydrocarbon core. The distribution of the water tends to mirror that of the peptide. Unlike peptide-free bilayers, 5% of the water penetrates the hydrocarbon, which is about 100-fold greater than expected. A quantitative thermodynamic analysis of the interfacial binding of the peptides suggests that (1) the hydrophobic interactions are 60-70% complete upon binding at the bilayer interface, (2) the interface is likely to play an important role in helix formation and insertion, (3) the hydrogen bond status of amino acid side chains is crucial to insertion, and (4) an a priori lack of knowledge of the status of such bonds could limit the precision of hydrophobicity plots. We introduce an interfacial hydrophobicity scale, IFH(h), with a variable hydrogen bond parameter (h) that permits one to consider explicitly hydrogen bonding in transbilayer helix searches.     

Differential effect of amino acid residues on the stability of double helices formed from polyribonucleotides and its possible relation to the evolution of the genetic code

Summary The interaction of amino acid residues with polyribonucleotides was characterized by measurements of melting temperatures (t_m) for poly(A) poly(U) and poly(I)poly(C) as functions of the concentrations of various amino acid amides. The amides of hydrophilic amino acids lead to a continuous increase of tm with increasing concentration, whereas amides of hydrophobic amino acids induce a decrease of t_m at low concentrations (1 mM) followed by an increase at higher concentrations. Analysis of the data by a simple site model provides the affinity of each ligand for the double helix relative to that for the single strands. This parameter decreases in the order Ala>Gly>Ser>Asn>Pro>Met, Val>Ile, Leu for poly(A) poly(U) and Ala, Gly, Ser>Asn>Pro>Val>Ile, Met, Leu for poly(I)poly(C). The special effects of hydrophobic amino acids may be related to the similarity of the codons for these amino acids. A simple model for assignment of codons to amino acids is proposed.     

Flavour formation by amino acid catabolism

Microbial catabolism of amino acids produces flavour compounds of importance for foods such as cheese, wine and fermented sausages. Lactic acid bacteria are equipped with enzyme systems for using the amino acids in their metabolism and are useful for flavour formation of foods. Branched-chain amino acids (Leu, Ile, Val) are converted into compounds contributing to malty, fruity and sweaty flavours; catabolism of aromatic amino acids (Phe, Tyr, Trp) produce floral, chemical and faecal flavours; aspartic acid (Asp) is catabolised into buttery flavours and sulphuric amino acids (Met, Cys) are transferred into compounds contributing to boiled cabbage, meaty and garlic flavours.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Differential effect of amino acid residues on the stability of double helices formed from polyribonucleotides and its possible relation to the evolution of the genetic code

The interaction of amino acid residues with polyribonucleotides was characterized by measurements of melting temperatures (tm) for poly(A).poly(U) and poly(I).poly(C) as functions of the concentrations of various amino acid amides. The amides of hydrophilic amino acids lead to a continuous increase of tm with increasing concentration, whereas amides of hydrophobic amino acids induce a decrease of tm at low concentrations (approximately 1 mM) followed by an increase at higher concentrations. Analysis of the data by a simple site model provides the affinity of each ligand for the double helix relative to that for the single strands. This parameter decreases in the order Ala greater than Gly greater than Ser greater than Asn greater than Pro greater than Met, Val greater than Ile, Leu for poly(A).poly(U) and Ala, Gly, Ser greater than Asn greater than Pro greater than Val greater than Ile, Met, Leu for poly(I).poly(C). The special effects of hydrophobic amino acids may be related to the similarity of the codons for these amino acids. A simple model for assignment of codons to amino acids is proposed.     

癌肿与氨基酸代谢的研究

研究了癌肿与氨基酸代谢的关系。这些癌肿包括喉癌HepⅡ细胞 ,急性非淋巴细胞白血病和急性淋巴细胞白血病 ,结果表明 :( 1 )喉癌细胞株培养过程中亮氨酸、赖氨酸、丝氨酸、天冬酰胺、异亮氨酸、甘氨酸以及苏氨酸等水平明显降低 ,而色氨酸水平明显增加 ,说明喉癌细胞的生长繁殖必须依赖以上 7种氨基酸同时释放了色氨酸 ;( 2 )急性非淋巴细胞白血病 (ANLL)患者血浆中的谷氨酸、甘氨酸、亮氨酸、苯丙氨酸、酪氨酸和色氨酸等水平明显升高 ,而苏氨酸、组氨酸、丙氨酸等水平明显降低 ,这些结果与国际报道相一致 ;( 3)经治疗后 ,ANLL患者血浆中甘氨酸、色氨酸和苯丙氨酸等水平明显降低 ,而丙氨酸、组氨酸等水平明显升高 ,表明肿瘤细胞处在无氧代谢。患者经治疗后色氨酸和苯丙氨酸水平降低和组氨酸水平的升高对患者预后是有益的 ;( 4)急性淋巴细胞白血病患者血浆中苯丙氨酸、赖氨酸、色氨酸和酪氨酸水平提高 ,这些氨基酸能促进肿瘤生长 ,而门冬酰胺、谷氨酰胺以及天冬氨酸水平降低 ,说明这 3种氨基酸为肿瘤生长所必须。此外还发现ALL患者外周淋巴细胞中精氨酸水平增加 ,精氨酸对癌肿细胞有直接杀伤作用。     

Effect of multiple aliphatic amino acids substitutions on the structure, function, and mode of action of diastereomeric membrane active peptides

The initial stages leading to the binding and functioning of membrane-active polypeptides including hormones, signal sequences, and lytic peptides are mainly governed by electrostatic attraction and hydrophobic partitioning between water and lipid bilayers. Antimicrobial peptides serve as an important model for studying the details of these initial steps. However, a systematic analysis of the contribution of multiple hydrophobic amino acids to these steps have been hindered by the propensity of many peptides to aggregate and become inactivated in solution. To this end, we synthesized a series of model amphipathic all L-amino acid peptides and their diastereomers with the sequence KX(3)KWX(2)KX(2)K, where X = Gly, Ala, Val, Ile, or Leu. The effect of the aliphatic amino acids on the biological activity, binding, structure, membrane localization, and mode of action of these peptides was investigated. Most of the L-amino acid peptides oligomerized and adopted distinct structures in solution and in a membrane mimetic environment. Among this group only the Leu containing peptide was hemolytic and highly active on most bacteria tested. The Val- and Leu-containing peptides were hemolytic but inactive toward most bacteria tested. In contrast, the diastereomeric peptides were monomeric and unstructured in solution, but they adopted distinct structures upon membrane binding. While hemolytic activity was drastically reduced, the spectrum of antibacterial activity was preserved or increased. Importantly, we found a direct correlation with the diastereomers between hydrophobicity and propensity to form a helical/distorted-helix and activity (induced membrane leakage and antibacterial activity), despite the fact that they contained 30% D-amino acids. Furthermore, efficient increase in membrane permeability can proceed through different mechanisms. Specifically, the Leu-containing diastereomeric peptide micellized vesicles and possibly bacterial membranes while the Ile-containing diastereomeric peptide fused model membranes and irregularly disrupted bacterial membranes.     

Structure-based statistical analysis of transmembrane helices

Recent advances in determination of the high-resolution structure of membrane proteins now enable analysis of the main features of amino acids in transmembrane (TM) segments in comparison with amino acids in water-soluble helices. In this work, we conducted a large-scale analysis of the prevalent locations of amino acids by using a data set of 170 structures of integral membrane proteins obtained from the MPtopo database and 930 structures of water-soluble helical proteins obtained from the protein data bank. Large hydrophobic amino acids (Leu, Val, Ile, and Phe) plus Gly were clearly prevalent in TM helices whereas polar amino acids (Glu, Lys, Asp, Arg, and Gln) were less frequent in this type of helix. The distribution of amino acids along TM helices was also examined. As expected, hydrophobic and slightly polar amino acids are commonly found in the hydrophobic core of the membrane whereas aromatic (Trp and Tyr), Pro, and the hydrophilic amino acids (Asn, His, and Gln) occur more frequently in the interface regions. Charged amino acids are also statistically prevalent outside the hydrophobic core of the membrane, and whereas acidic amino acids are frequently found at both cytoplasmic and extra-cytoplasmic interfaces, basic amino acids cluster at the cytoplasmic interface. These results strongly support the experimentally demonstrated biased distribution of positively charged amino acids (that is, the so-called the positive-inside rule) with structural data.     

Relative spatial positions of tryptophan and cationic residues in helical membrane-active peptides determine their cytotoxicity

The cytotoxic activity of 10 analogs of the idealized amphipathic helical 21-mer peptide (KAAKKAA)3, where three of the Ala residues at different positions have been replaced with Trp residues, has been investigated. The peptide's cytotoxic activity was found to be markedly dependent upon the position of the Trp residues within the hydrophobic sector of an idealized α-helix. The peptides with Trp residues located opposite the cationic sector displayed no antitumor activity, whereas those peptides with two or three Trp residues located adjacent to the cationic sector exhibited high cytotoxic activity when tested against three different cancer cell lines. Dye release experiments revealed that in contrast to the peptides with Trp residues located opposite the cationic sector, the peptides with Trp residues located adjacent to the cationic sector induced a strong permeabilizing activity from liposomes composed of a mixture of zwitterionic phosphatidylcholine and negatively charged phosphatidylserine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS)) (2:1) but not from liposomes composed of zwitterionic phosphatidylcholine, POPC. Fluorescence blue shift and quenching experiments revealed that Trp residues inserted deeper into the hydrophobic environment of POPC/POPS liposomes for peptides with high cytotoxic activity. Through circular dichroism studies, a correlation between the cytotoxic activity and the α-helical propensity was established. Structural studies of one inactive and two active peptides in the presence of micelles using NMR spectroscopy showed that only the active peptides adopted highly coiled to helical structures when bound to a membrane surface.     

Packing and hydrophobicity effects on protein folding and stability: effects of beta-branched amino acids, valine and isoleucine, on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers.

The aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three-dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA-binding proteins (leucine zippers), Leu residues are conserved at positions "d," and beta-branched amino acids, Ile and Val, often occur at positions "a" in the hydrophobic core. In order to discern what effect this selective distribution of hydrophobes has on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers, three Val or three Ile residues were simultaneously substituted for Leu at either positions "a" (9, 16, and 23) or "d" (12, 19, and 26) in both chains of a model coiled coil. The stability of the resulting coiled coils was monitored by CD in the presence of Gdn.HCl. The results of the mutations of Ile to Val at either positions "a" or "d" in the reduced or oxidized coiled coils showed a significant hydrophobic effect with the additional methylene group in Ile stabilizing the coiled coil (delta delta G values range from 0.45 to 0.88 kcal/mol/mutation). The results of mutations of Leu to Ile or Val at positions "a" in the reduced or oxidized coiled coils showed a significant packing effect in stabilizing the coiled coil (delta delta G values range from 0.59 to 1.03 kcal/mol/mutation). Our results also indicate the subtle control hydrophobic packing can have not only on protein stability but on the conformation adopted by the amphipathic alpha-helices. These structural findings correlate with the observation that in DNA-binding proteins, the conserved Leu residues at positions "d" are generally less tolerant of amino acid substitutions than the hydrophobic residues at positions "a."     

Large alkyl side-chains of isoleucine and leucine in the NPIRL region constitute the core of the vacuolar sorting determinant of sporamin precursor

The N-terminal propeptide of the sporamin precursor contains vacuolar targeting information within the Asn-26/Pro-27/Ile-28/Arg-29/Leu-30 (NPIRL) sequence. An Agrobacterium-mediated transient expression assay with tobacco BY-2 cells was employed to investigate the role of each amino acid of the NPIRL region in vacuolar targeting. Replacement of Asn-26, Pro-27, Ile-28 and Leu-30 with several amino acids caused secretion of the mutant prosporamin. Leu was the only amino acid that could be substituted for Ile-28 without affecting transport. Exchange of Leu-30 for amino acids with small side-chains abolished vacuolar delivery. These results indicate that the consensus composition of the NPIRL sequence is [preferably Asn]-[not acidic]-[Ile or Leu]-[any amino acid]-[large and hydrophobic] and suggest that the large alkyl side-chains of Ile-28 and Leu-30 constitute the core of the vacuolar sorting determinant.     

Prebiotic synthesis and organization of biopolymer-like macromolecules

Prebiotic synthesis of bio-oligomers containing more than five units are examined with respect to chemical divergence and physical dispersion of the products. Experiments are presented which support a possible pathway running from a primitive mixture of amino acids to peptides organized into stereospecific -sheet surfaces. This pathway includes selective condensation of amino acids through the racemization-free N-carboxyanhydride polymerization, specific aggregation of alternating sequences with hydrophilic and hydrophobic residues and enantioselection of homochiral sequences.     

Highly restricted distributions of hydrophobic and charged amino acids in longitudinal quadrants of alpha-helices

Helix formation in folding proteins is stabilized by binding of recurrent hydrophobic side chains in one longitudinal quadrant against the locally most hydrophobic region of the protein. To test this hypothesis, we fitted sequences of 247 alpha-helices of 55 proteins to the circular (infinite) template (symbol; see text) to maximize the strip-of-helix hydrophobicity index (the mean hydrophobicity of residues in (symbol; see text) positions). These template-predicted configurations closely matched crystallographic structures in 87% of four- or five-turn helices compared. We determined the longitudinal quadrant distributions of amino acids in the template-fitted, sheet projections of alpha-helices with respect to the best longitudinal, hydrophobic strip on each helix and to the N and C termini, interiors, and entire helices. Amino acids Leu, Ile, Val, and Phe were concentrated in one longitudinal quadrant (p less than 0.001). Lys, Arg, Asp, and Glu were not in the quadrant of Leu, Ile, Val, and Phe (p less than 0.001). Significant quadrant distributions for other amino acids and for termini of the helices were also found.     

Identification of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase.

Smooth muscle myosin light chain kinase (smMLCK) is a Ca(2+)-calmodulin (CaM)-dependent enzyme that phosphorylates the 20-kDa light chains of myosin. In a previous study (Bagchi, I.C., Kemp, B.E., and Means, A.R. (1989) J. Biol. Chem. 264, 15843-15849), we expressed in bacteria a 40-kDa fragment of smMLCK that displayed Ca(2+)-CaM-regulated catalytic activity. Initial mutagenesis experiments indicated that Gly811 and Arg812 were important for CaM-dependent activation of this 40-kDa enzyme. We have now carried out site-directed mutagenesis within the CaM-binding domain (Ser787 to Leu813) of this enzyme to identify amino acids that are critical for CaM binding and activation. Our studies reveal that the individual mutation of several hydrophobic amino acid residues such as Leu813, Ile810, and Trp800 and the glycine residue Gly804 also resulted in a severe decrease in or complete loss of CaM binding and activation of smMLCK. The hydrophobic residue (Trp800) and the basic residue (Arg812), both of which are mandatory for CaM binding to smMLCK, occur in analogous positions within the CaM-binding domain of a number of CaM-regulated enzymes. We conclude from these results that CaM binding by smMLCK is determined by an interplay of specific hydrophobic and electrostatic interactions which appear to be conserved among various target enzymes of CaM.     

The role of leucine and isoleucine in tuning the hydropathy of class A GPCRs

Leucine and Isoleucine are two amino acids that differ only by the positioning of one methyl group. This small difference can have important consequences in α-helices, as the β-branching of Ile results in helix destabilization. We set out to investigate whether there are general trends for the occurrences of Leu and Ile residues in the structures and sequences of class A GPCRs (G protein-coupled receptors). GPCRs are integral membrane proteins in which α-helices span the plasma membrane seven times and which play a crucial role in signal transmission. We found that Leu side chains are generally more exposed at the protein surface than Ile side chains. We explored whether this difference might be attributed to different functions of the two amino acids and tested if Leu tunes the hydrophobicity of the transmembrane domain based on the Wimley-White whole-residue hydrophobicity scales. Leu content decreases the variation in hydropathy between receptors and correlates with the non-Leu receptor hydropathy. Both measures indicate that hydropathy is tuned by Leu. To test this idea further, we generated protein sequences with random amino acid compositions using a simple numerical model, in which hydropathy was tuned by adjusting the number of Leu residues. The model was able to replicate the observations made with class A GPCR sequences. We speculate that the hydropathy of transmembrane domains of class A GPCRs is tuned by Leu (and to some lesser degree by Lys and Val) to facilitate correct insertion into membranes and/or to stably anchor the receptors within membranes.