Statistical analysis of predicted transmembrane alpha-helices

Statistical analyses were undertaken for putative transmembrane alpha-helices obtained from a database representing the subset of membrane proteins available in Swiss-Prot. The average length of a transmembrane alpha-helix was found to be 22-21 amino acids with a large variation around the mean. The transfer free energy from water to oil of a transmembrane alpha-helix in bitopic proteins, -48 kcal/mol, is higher than that in polytopic proteins, -39 kcal/mol, and is nearly identical to that obtained by assuming a random distribution of solely hydrophobic amino acids in the alpha-helix. The amino acid composition of hydrophobic residues is similar in bitopic and polytopic proteins. In contrast, the more polar the amino acids are, the less likely they are to be found in bitopic proteins compared to polytopic ones. This most likely reflects the ability of alpha-helical bundles to shield the polarity of residues from the hydrophobic bilayer. One half of all amino acids were distributed nonrandomly in both bitopic and polytopic proteins. A preference was found for tyrosine and tryptophan residues to be at the ends of transmembrane alpha-helices. Correlated distribution analysis of amino acid pairs indicated that most amino acids are independently distributed in each helix. Exceptions are cysteine, tyrosine, and tryptophan which appear to cluster closely to one another and glycines which are preferentially found on the same side of alpha-helices.     

Energetic cost and structural consequences of burying a hydroxyl group within the core of a protein determined from Ala-->Ser and Val-->Thr substitutions in T4 lysozyme

In order to determine the thermodynamic cost of introducing a polar group within the core of a protein, a series of nine Ala-->Ser and 3 Val-->Thr substitutions was constructed in T4 lysozyme. The sites were all within alpha-helices but ranged from fully solvent-exposed to totally buried. The range of destabilization incurred by the Ala-->Ser substitutions was found to be very similar to that for the Val-->Thr replacements. For the solvent-exposed and partly exposed sites the destabilization was modest (approximately less than 0.5 kcal/mol). For the completely buried sites the destabilization was larger, but variable (approximately 1-3 kcal/mol). Crystal structure determinations showed that the Ala-->Ser mutant structures were, in general, very similar to their wild-type counterparts, even though the replacements introduce a hydroxyl group. This is in part because the introduced serines are all within alpha-helices and at congested sites can avoid steric clashes with surrounding atoms by making a hydrogen bond to a backbone carbonyl oxygen in the preceding turn of the helix. The three substituted threonine side chains essentially superimpose on their valine counterparts but display somewhat larger conformational adjustments. The results illustrate how a protein structure will adapt in different ways to avoid the presence of an unsatisfied hydrogen bond donor or acceptor. In the most extreme case, Val 149-->Thr, which is also the most destabilizing variant (delta delta G = 2.8 kcal/mol), a water molecule is incorporated in the mutant structure in order to provide a hydrogen-bonding partner. The results are consistent with the view that many hydrogen bonds within proteins contribute only marginally to stability but that noncharged polar groups that lack a hydrogen-bonding partner are very destabilizing (delta delta G approximately greater than 3 kcal/mol). Supportive of other studies, the alpha-helix propensity of alanine is seen to be higher than that of serine (delta delta G = 0.46 +/- 0.04 kcal/mol), while threonine and valine are similar in alpha-helix propensity.     

Relationship of sidechain hydrophobicity and alpha-helical propensity on the stability of the single-stranded amphipathic alpha-helix

The aim of the present investigation is to determine the effect of alpha-helical propensity and sidechain hydrophobicity on the stability of amphipathic alpha-helices. Accordingly, a series of 18-residue amphipathic alpha-helical peptides has been synthesized as a model system where all 20 amino acid residues were substituted on the hydrophobic face of the amphipathic alpha-helix. In these experiments, all three parameters (sidechain hydrophobicity, alpha-helical propensity and helix stability) were measured on the same set of peptide analogues. For these peptide analogues that differ by only one amino acid residue, there was a 0.96 kcal/mole difference in alpha-helical propensity between the most (Ala) and the least (Gly) alpha-helical analogue, a 12.1-minute difference between the most (Phe) and the least (Asp) retentive analogue on the reversed-phase column, and a 32.3 degrees C difference in melting temperatures between the most (Leu) and the least (Asp) stable analogue. The results show that the hydrophobicity and alpha-helical propensity of an amino acid sidechain are not correlated with each other, but each contributes to the stability of the amphipathic alpha-helix. More importantly, the combined effects of alpha-helical propensity and sidechain hydrophobicity at a ratio of about 2:1 had optimal correlation with alpha-helix stability. These results suggest that both alpha-helical propensity and sidechain hydrophobicity should be taken into consideration in the design of alpha-helical proteins with the desired stability.     

pi-Stacking interactions. Alive and well in proteins

A representative set of high resolution x-ray crystal structures of nonhomologous proteins have been examined to determine the preferred positions and orientations of noncovalent interactions between the aromatic side chains of the amino acids phenylalanine, tyrosine, histidine, and tryptophan. To study the primary interactions between aromatic amino acids, care has been taken to examine only isolated pairs (dimers) of amino acids because trimers and higher order clusters of aromatic amino acids behave differently than their dimer counterparts. We find that pairs (dimers) of aromatic side chain amino acids preferentially align their respective aromatic rings in an off-centered parallel orientation. Further, we find that this parallel-displaced structure is 0.5-0.75 kcal/mol more stable than a T-shaped structure for phenylalanine interactions and 1 kcal/mol more stable than a T-shaped structure for the full set of aromatic side chain amino acids. This experimentally determined structure and energy difference is consistent with ab initio and molecular mechanics calculations of benzene dimer, however, the results are not in agreement with previously published analyses of aromatic amino acids in proteins. The preferred orientation is referred to as parallel displaced pi-stacking.     

Stabilizing the subtilisin BPN' pro-domain by phage display selection: how restrictive is the amino acid code for maximum protein stability?

We have devised a procedure using monovalent phage display to select for stable mutants in the pro-domain of the serine protease, subtilisin BPN'. In complex with subtilisin, the pro-domain assumes a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helices. When isolated, however, the pro-domain is 97% unfolded. These experiments use combinatorial mutagenesis to select for stabilizing amino acid combinations at a particular structural locus and determine how many combinations are close to the maximum protein stability. The selection for stability is based on the fact that the independent stability of the pro-domain is very low and that binding to subtilisin is thermodynamically linked to folding. Two libraries of mutant pro-domains were constructed and analyzed to determine how many combinations of amino acids at a particular structural locus result in the maximum stability. A library comprises all combinations of four amino acids at a structural locus. Previous studies using combinatorial genetics have shown that many different combinations of amino acids can be accommodated in a selected locus without destroying function. The present results indicate that the number of sequence combinations at a structural locus, which are close to the maximum stability, is small. The most striking example is a selection at an interior locus of the pro-domain. After two rounds of phagemid selection, one amino acid combination is found in 40% of sequenced mutants. The most frequently selected mutant has a deltaG(unfolding) = 4 kcal/mol at 25 degrees C, an increase of 6 kcal/mol relative to the naturally occurring sequence. Some implications of these results on the amount of sequence information needed to specify a unique tertiary fold are discussed. Apart from possible implications on the folding code, the phage display selection described here should be useful in optimizing the stability of other proteins, which can be displayed on the phage surface.     

Geometrical and sequence characteristics of alpha-helices in globular proteins

Understanding the sequence-structure relationships in globular proteins is important for reliable protein structure prediction and de novo design. Using a database of 1131 alpha-helices with nonidentical sequences from 205 nonhomologous globular protein chains, we have analyzed structural and sequence characteristics of alpha-helices. We find that geometries of more than 99% of all the alpha-helices can be simply characterised as being linear, curved, or kinked. Only a small number of alpha-helices ( approximately 4%) show sharp localized bends in their middle regions, and thus are classified as kinked. Approximately three-fourths (approximately 73%) of the alpha-helices in globular proteins show varying degrees of smooth curvature, with a mean radius of curvature of 65 +/- 33 A; longer helices are less curved. Computation of helix accessibility to the solvent indicates that nearly two-thirds of the helices ( approximately 66%) are largely buried in the protein core, and the length and geometry of the helices are not correlated with their location in the protein globule. However, the amino acid compositions and propensities of individual amino acids to occur in alpha-helices vary with their location in the protein globule, their geometries, and their lengths. In particular, Gln, Glu, Lys, and Arg are found more often in helices near the surface of globular proteins. Interestingly, kinks often seem to occur in regions where amino acids with low helix propensities (e.g., beta-branched and aromatic residues) cluster together, in addition to those associated with the occurrence of proline residues. Hence the propensities of individual amino acids to occur in a given secondary structure depend not only on conformation but also on its length, geometry, and location in the protein globule.     

Thermodynamic analysis of human plasma apolipoprotein C-1: high-temperature unfolding and low-temperature oligomer dissociation

Thermal and chemical unfolding of lipid-free apolipoprotein C-1 (apoC-1), a 6-kDa protein component of very low density and high-density lipoproteins, was analyzed by far-UV CD. In neutral 1 mM Na2HPO4 solutions containing 6-7 micrograms/mL protein, the apoC-1 monomer is approximately 30% alpha-helical at 0-22 degrees C and unfolds reversibly from about 22-80 degrees C with Tm = 51 +/- 3 degrees C and van't Hoff enthalpy delta Hv(Tm) = 19 +/- 3 kcal/mol. The apparent free energy of the monomer stabilization determined from the chemical unfolding at 0 degree C, delta G(0 degree C) = 2.8 +/- 0.8 kcal/mol, decreases by about 1 kcal/mol upon heating to 25 degrees C. A small apparent heat capacity increment suggests the absence of a substantial hydrophobic core for the apoC-1 molecule. At pH 7, increasing apoC-1 concentration above 10 micrograms/mL leads to self-association and formation of additional alpha-helices that unfold upon both heating and cooling from room temperature. The CD data indicate that the high-temperature transition reflects a complete monomer unfolding and the low-temperature transition reflects oligomer dissociation into stable monomers. This suggests the importance of hydrophobic interactions for apoC-1 self-association. Close proximity between the high- and low-temperature transitions and the absence of a plateau in the chemical unfolding curves recorded from oligomeric apoC-1 indicate marginal oligomer stability and suggest that in vivo apoC-1 transfer is mediated via the complexes with other apolipoproteins and/or lipids.     

Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation

To prevent the loss of raw material in ethanol production by anaerobic yeast cultures, glycerol formation has to be reduced. In theory, this may be done by providing the yeast with amino acids, since the de novo cell synthesis of amino acids from glucose and ammonia gives rise to a surplus of NADH, which has to be reoxidized by the formation of glycerol. An industrial strain of Saccharomyces cerevisiae was cultivated in batch cultures with different nitrogen sources, i.e., ammonium salt, glutamic acid, and a mixture of amino acids, with 20 g of glucose per liter as the carbon and energy source. The effects of the nitrogen source on metabolite formation, growth, and cell composition were measured. The glycerol yields obtained with glutamic acid (0.17 mol/mol of glucose) or with the mixture of amino acids (0.10 mol/mol) as a nitrogen source were clearly lower than those for ammonium-grown cultures (0.21 mol/mol). In addition, the ethanol yield increased for growth on both glutamic acid (by 9%) and the mixture of amino acids (by 14%). Glutamic acid has a large influence on the formation of products; the production of, for example, alpha-ketoglutaric acid, succinic acid, and acetic acid, increased compared with their production with the other nitrogen sources. Cultures grown on amino acids have a higher specific growth rate (0.52 h-1) than cultures of both ammonium-grown (0.45 h-1) and glutamic acid-grown (0.33 h-1) cells. Although the product yields differed, similar compositions of the cells were attained. The NADH produced in the amino acid, RNA, and extracellular metabolite syntheses was calculated together with the corresponding glycerol formation. The lower-range values of the theoretically calculated yields of glycerol were in good agreement with the experimental yields, which may indicate that the regulation of metabolism succeeds in the most efficient balancing of the redox potential.     

The evolution of proteins from random amino acid sequences. I. Evidence from the lengthwise distribution of amino acids in modern protein sequences

We examine in this paper one of the expected consequences of the hypothesis that modern proteins evolved from random heteropeptide sequences. Specifically, we investigate the lengthwise distributions of amino acids in a set of 1,789 protein sequences with little sequence identify using the run test statistic (ro) of Mood (1940, Ann. Math. Stat. 11, 367-392). The probability density of ro for a collection of random sequences has mean = 0 and variance = 1 [the N(0,1) distribution] and can be used to measure the tendency of amino acids of a given type to cluster together in a sequence relative to that of a random sequence. We implement the run test using binary representations of protein sequences in which the amino acids of interest are assigned a value of 1 and all others a value of 0. We consider individual amino acids and sets of various combinations of them based upon hydrophobicity (4 sets), charge (3 sets), volume (4 sets), and secondary structure propensity (3 sets). We find that any sequence chosen randomly has a 90% or greater chance of having a lengthwise distribution of amino acids that is indistinguishable from the random expectation regardless of amino acid type. We regard this as strong support for the random-origin hypothesis. However, we do observe significant deviations from the random expectation as might be expected after billions years of evolution. Two important global trends are found: (1) Amino acids with a strong alpha-helix propensity show a strong tendency to cluster whereas those with beta-sheet or reverse-turn propensity do not. (2) Clustered rather than evenly distributed patterns tend to be preferred by the individual amino acids and this is particularly so for methionine. Finally, we consider the problem of reconciling the random nature of protein sequences with structurally meaningful periodic "patterns" that can be detected by sliding-window, autocorrelation, and Fourier analyses. Two examples, rhodopsin and bacteriorhodopsin, show that such patterns are a natural feature of random sequences.     

Measurement of the beta-sheet-forming propensities of amino acids

Several model systems have been used to evaluate the alpha-helical propensities of different amino acids. In contrast, experimental quantitation of beta-sheet preferences has been addressed in only one model system, a zinc-finger peptide. Here we measure the relative propensity for beta-sheet formation of the twenty naturally occurring amino acids in a variant of the small, monomeric, beta-sheet-rich, IgG-binding domain from protein G. Amino-acid substitutions were made at a guest site on the solvent-exposed surface of the beta-sheet. Several criteria were used to establish that the mutations did not cause significant structural changes: binding to the Fc domain of IgG, calorimetric unfolding and NMR spectroscopy. Characterization of the terminal stabilities of these proteins leads to a thermodynamic scale for beta-sheet propensities that spans a range of approximately 2 kcal mol-1 for the naturally occurring amino acids, excluding proline. The magnitude of the differences suggests that beta-sheet preferences can be important determinants of protein stability.     

Age dependence of total cerebral blood flow volume from childhood to adulthood

Three-dimensional structures of a representative set of more than 30 hydrogen-bonded nucleic acids pairs have been studied by reliable ab initio quantum mechanical methods. We show that many hydrogen-bonded nucleic acid base pairs are intrinsically nonplanar, mainly due to the partial sp3 hybridization of nitrogen atoms of their amino groups and secondary electrostatic interactions. This finding extends the variability of intermolecular interactions of DNA bases in that i) flexibility of the base pairs is larger than has been assumed before, and ii) attractive proton-proton acceptor interactions oriented out of the base pair plane are allowed. For example, all four G-A mismatch base pairs are propeller twisted, and the energy preferences for the nonplanar structures range from less than 0.1 kcal/mol to 1.8 kcal/mol. We predict that nonplanarity of the amino group of guanine in the G(anti)...A(anti) pair of the ApG step of the d(CCAAGATTGG)2 crystal structure is an important stabilizing factor that improves the energy of this structure by almost 3 kcal/mol. Currently used empirical potentials are not accurate enough to properly cover the interactions associated with amino-group and base-pair nonplanarity.     

Helix-capping interaction in lambda Cro protein: a free energy simulation analysis

The stability mutant Tyr-26-->Asp was studied in the Cro protein from bacteriophage lambda using free energy molecular dynamics simulations. The mutant was calculated to be more stable than the wild type by 3.0 +/- 1.7 kcal/mol/monomer, in reasonable agreement with experiment (1.4 kcal/mol/monomer). Moreover, the aspartic acid in the mutant was found to form a capping interaction with the amino terminus of the third alpha-helix of Cro. The simulations were analyzed to understand better the source of the stability of this helix-capping interaction and to examine the results in light of previous explanations of stabilizing helix caps--namely, a model of local unsatisfied hydrogen bonds at the helix termini and the helix macrodipole model. Analysis of the simulations shows that the stabilizing effect of this charged helical cap is due both to favorable hydrogen bonds with backbone NH groups at the helix terminus and to favorable electrostatic interactions (but not hydrogen bonds) with their carbonyls (effectively the next row of local dipoles in the helix). However, electrostatic interactions are weak or negligible with backbone dipolar groups in the helix further away from the terminus. Moreover, the importance of other local electrostatic interactions with polar side chains near the helix terminus, which are neglected in most treatments of this effect, are shown to be important. Thus, the results support a model that is intermediate between the two previous explanations: both unsatisfied hydrogen bonds at the helix terminus and other, local preoriented dipolar groups stabilize the helix cap. These findings suggest that similar interactions with preoriented dipolar groups may be important for cooperativity in other charge-dipole interactions and may be employed to advantage for molecular design.     

Solution conformations and thermodynamics of structured peptides: molecular dynamics simulation with an implicit solvation model

Calculations of the ensemble of solution conformations and thermodynamics of an analogue of the C-terminal helix of ribonuclease A (RN24) and of a synthetic, beta-hairpin forming peptide (BH8) are presented. For efficient sampling of conformation space, molecular dynamics simulations with an implicit solvent potential and umbrella sampling of the potential energy are performed. Starting from the fully extended chains, the simulations yield several folding and unfolding transitions between disordered (coil) conformations of the peptides and the "native" state (RN24, helix; BH8, hairpin); the simulations also lead to the occurrence of "misfolded" conformations (RN24, hairpin; BH8, helix). In agreement with experiment, the calculations predict 58% helix for RN24 at 275 K and an antiparallel-beta content of 38% at 275 K for BH8; the calculated probabilities for the misfolded species are 2% or smaller at all temperatures considered (250-1100 K). Good agreement is also shown between the calculated 3JHNalpha spin-spin coupling constants of RN24 and BH8 at 275 K, and those obtained from NMR experiments at the same temperature. From the calculated probabilities of helix (h), beta-hairpin (b), and coil (c), the free energy differences between the structured substates are DeltaGch=Gc-Gh approximately 1 kcal/mol and DeltaGbh>/=1.8 kcal/mol for RN24, and DeltaGcb approximately 0.7 kcal/mol and DeltaGhb>/=2.7 kcal/mol for BH8. The free energy difference between "correctly" folded and misfolded secondary structures are of interest for understanding the alpha to beta transition that is thought to play a role in amyloid fibril formation.     

Surface salt bridges stabilize the GCN4 leucine zipper

We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled-coil. Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites. At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges. Introduction of three alanines stabilizes the dimer by 1.1 kcal/mol relative to the wild-type. An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1.7 kcal/ mol, an apparent elevation of the melting temperature relative to wild type of about 22 degrees C. While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles. Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled-coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences.     

The MHC of a broiler chicken line: serology, B-G genotypes, and B-F/B-LB sequences

The tumor suppresser protein p53 has been called the "guardian of the genome." DNA damage induces p53 to either halt the cell cycle, allowing for repair, or initiate apoptosis. P53 is mutated in over 50% of human tumors and it has been proposed that many tumorigenic mutations are deleterious to p53 because they induce local unfolding. To explore this hypothesis, peptide models have been developed to study tumorigenic mutations in the H2 helix of the p53 core domain. This helix is rich with charged residues and is a key component of the DNA binding region. A 16-residue peptide corresponding to the H2 wild-type sequence extended with an Ala-rich C-terminus was synthesized and studied by 1H-nmr (500 MHz) and CD. The nmr studies demonstrate that this peptide adopts helical structure in solution. Six additional peptides corresponding to subtle tumorigenic mutations were synthesized and CD was used to assess the relative stability of these "mutant analogues." All six mutations studied are destabilizing relative to the wild type, with delta delta G values in the range of 0.26 to 1.35 kcal mol-1. Surprisingly, substitution of Asp 281 with Ala resulted in a peptide with the greatest destabilization even though Ala possesses the largest helix propensity of the common 20 amino acids. Because this helix appears to be stabilized mainly by local electrostatics, we conclude that its structure is susceptible to even the most conservative mutations. These results provide support for the hypothesis that tumorigenic mutations induce local unfolding of p53.     

Stereochemical punctuation marks in protein structures: glycine and proline containing helix stop signals

An analysis on the nature of alpha-helix stop signals has been carried out, using a dataset of 1057 helices identified from 250 high resolution (相似文献   

Effect of peptide binding on amide proton exchange rates in the PDZ2 domain from human phosphatase hPTP1E

Amide hydrogen-deuterium exchange rates were measured in the PDZ2 domain from human phosphatase hPTPIE by 1H-15N heteronuclear NMR spectroscopy. Protection factors were calculated for the slowly exchanging hydrogens in both the free PDZ2 domain and its complex with an octapeptide peptide, R-N-E-I-Q-S-L-V, derived from the C-terminus of the Fas receptor. Aside from a short alpha-helical region alpha1 (amino acids A-45 to D-49), the pattern of highly protected amides correlated well with the presence of hydrogen bonds in elements of the secondary structure. Hydrogen-bonded amides showed relatively fast exchange rates with half-lives of less than 9 h at pD 7.6 and 8 degrees C. Protection factors, calculated as the ratio of theoretical (denatured) and observed exchange rates, showed less dispersion in maximal values than did the actual exchange rates. This behavior and the large pH dependence of the exchange rates suggest that amide exchange is close to the EX2 limit. In this limit, exchange of the most protected amides occurs through a global unfolding mechanism. The free energy of the unfolding calculated from the largest protection factors is 4.8 +/- 0.4 kcal/mol (1 cal = 4.184 J). This deltaG(o) closely matches the value measured by experiments with guanidine hydrochloride and fluorescence emission spectroscopy. Peptide binding to PDZ2 resulted in mostly global effects and stabilized the folded domain by 1.4 kcal/mol.     

Helix propensities are identical in proteins and peptides

Our understanding of the factors stabilizing alpha-helical structure has been greatly enhanced by the study of model alpha-helical peptides. However, the relationship of these results to the folding of helices in intact proteins is not well characterized. Helix propensities measured in model peptides are not in good agreement with those from proteins. In order to address these questions, we have measured helix propensities in the alpha-helix of ribonuclease T1 and a helical peptide of identical sequence. We have previously demonstrated excellent agreement between peptide and protein for the nonpolar amino acids [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2833-2837]. Most other amino acids also show good agreement, although certain polar amino acids are exceptions. Helix propensities measured in the ribonuclease T1 peptide/protein are compared with those measured in other systems. Reasonable agreement is found between most systems; however, our propensities differ substantially from those measured in several model peptide systems. Alanine-based peptides overestimate the propensity differences by a factor of 2, and host/guest experiments underestimate them by a factor of 2-3.     

Solution structure of recombinant human interleukin-6

Interleukin-6 (IL-6) is a 185 amino acid cytokine which exerts multiple biological effects in vivo and whose dysregulation underlies several disease processes. The solution structure of recombinant human interleukin-6 has now been determined using heteronuclear three and four-dimensional NMR spectroscopy. The structure of the molecule was determined using 3044 distance and torsion restraints derived by NMR spectroscopy to generate an ensemble of 32 structures using a combined distance geometry/simulated annealing protocol. The protein contains five alpha-helices interspersed with variable-length loops; four of these helices constitute a classical four-helix bundle with the fifth helix located in the CD loop. There were no distance violations greater than 0.3 A in any of the final 32 structures and the ensemble has an average-to-the-mean backbone root-mean-square deviation of 0.50 A for the core four-helix bundle. Although the amino-terminal 19 amino acids are disordered in solution, the remainder of the molecule has a well defined structure that shares many features displayed by other long-chain four-helix bundle cytokines. The high-resolution NMR structure of hIL-6 is used to rationalize available mutagenesis data in terms of a heteromeric receptor complex.