Reduction-driven polypeptide folding by the delta Tt mechanism.

Poly(Gly-Val-Gly-Val-Pro), i.e., poly(GVGVP), exhibits composition and solute dependence of Tt, the temperature of the inverse temperature transition at which hydrophobic folding and assembly occur on raising the temperature. Importantly, a means whereby the value of Tt is lowered from above to below the working temperature becomes an isothermal means of driving folding and assembly, i.e., of achieving free energy transduction. Using poly[0.73(GVGVP),0.27(GK[NMeN]GVP)] where [NMeN] indicates N-methyl nicotinamide attached to the epsilon-NH2 of the Lys(K) residue, chemical and electrochemical reductions are found to remarkably lower the value of Tt; reduction can drive hydrophobic folding and assembly as effectively as decreasing ionization. Changing the redox state of a protein becomes yet another means of achieving free energy transduction by the delta Tt mechanism.     

Engineering protein-based machines to emulate key steps of metabolism (biological energy conversion)

Metabolism is the conversion of available energy sources to those energy forms required for sustaining and propagating living organisms; this is simply biological energy conversion. Proteins are the machines of metabolism; they are the engines of motility and the other machines that interconvert energy forms not involving motion. Accordingly, metabolic engineering becomes the use of natural protein-based machines for the good of society. In addition, metabolic engineering can utilize the principles, whereby proteins function, to design new protein-based machines to fulfill roles for society that proteins have never been called upon throughout evolution to fulfill. This article presents arguments for a universal mechanism whereby proteins perform their diverse energy conversions; it begins with background information, and then asserts a set of five axioms for protein folding, assembly, and function and for protein engineering. The key process is the hydrophobic folding and assembly transition exhibited by properly balanced amphiphilic protein sequences. The fundamental molecular process is the competition for hydration between hydrophobic and polar, e.g., charged, residues. This competition determines Tt, the onset temperature for the hydrophobic folding and assembly transition, Nhh, the numbers of waters of hydrophobic hydration, and the pKa of ionizable functions. Reported acid-base titrations and pH dependence of microwave dielectric relaxation data simultaneously demonstrate the interdependence of Tt, Nhh and the pKa using a series of microbially prepared protein-based poly(30mers) with one glutamic acid residue per 30mer and with an increasing number of more hydrophobic phenylalanine residues replacing valine residues. Also, reduction of nicotinamides and flavins is shown to lower Tt, i.e., to increase hydrophobicity. Furthermore, the argument is presented, and related to an extended Henderson-Hasselbalch equation, wherein reduction of nicotinamides represents an increase in hydrophobicity and resulting hydrophobic-induced pKa shifts become the basis for understanding a primary energy conversion (proton transport) process of mitochondria. Copyright 1998 John Wiley & Sons, Inc.     

Effect of NaCl on the exothermic and endothermic components of the inverse temperature transition of a model elastin-like polymer

TMDSC data have been employed to observe the effect of NaCl on the inverse temperature transition of the model elastin-like polymer (GVGVP)251. NaCl causes a decrease in Tt and an increase in DeltaH. The increase in enthalpy appears both in the enthalpy related with the folding of the polymer and in the contribution associated with disruption of the structured water of hydrophobic hydration. It has been suggested that the presence of NaCl may cause a better formation of water structures surrounding the apolar polymer chains.     

Criteria for downhill protein folding: calorimetry, chevron plot, kinetic relaxation, and single-molecule radius of gyration in chain models with subdued degrees of cooperativity

Recent investigations of possible downhill folding of small proteins such as BBL have focused on the thermodynamics of non-two-state, "barrierless" folding/denaturation transitions. Downhill folding is noncooperative and thermodynamically "one-state," a phenomenon underpinned by a unimodal conformational distribution over chain properties such as enthalpy, hydrophobic exposure, and conformational dimension. In contrast, corresponding distributions for cooperative two-state folding are bimodal with well-separated population peaks. Using simplified atomic modeling of a three-helix bundle-in a scheme that accounts for hydrophobic interactions and hydrogen bonding-and coarse-grained C(alpha) models of four real proteins with various degrees of cooperativity, we evaluate the effectiveness of several observables at defining the underlying distribution. Bimodal distributions generally lead to sharper transitions, with a higher heat capacity peak at the transition midpoint, compared with unimodal distributions. However, the observation of a sigmoidal transition is not a reliable criterion for two-state behavior, and the heat capacity baselines, used to determine the van't Hoff and calorimetric enthalpies of the transition, can introduce ambiguity. Interestingly we find that, if the distribution of the single-molecule radius of gyration were available, it would permit discrimination between unimodal and bimodal underlying distributions. We investigate kinetic implications of thermodynamic noncooperativity using Langevin dynamics. Despite substantial chevron rollovers, the relaxation of the models considered is essentially single-exponential over an extended range of native stabilities. Consistent with experiments, significant deviations from single-exponential behavior occur only under strongly folding conditions.     

The effect of amino acid substitution on protein-folding and -unfolding transition studied by computer simulation

Protein-folding and -unfolding transitions were studied by the method of computer simulation. The protein was modeled as a two-dimensional lattice polymer. Various energy terms were assumed to be operative between units composing the polymer. But hydrophobic interactions were neglected explicitly. Both thermodynamic and kinetic quantities were obtained from the simulation, and from their temperature dependence in the transition zone characteristics of the conformational transition of proteins were discussed. Two amino acid substituted models, differing in the location of substitution, were studied and compared with the original in order to clarify the effect of substitution on conformational transition of proteins. The following conclusions were reached in this study: (1) The relaxation time of the slow mode, which reflects the overall folding and unfolding processes, shows a peak near the transition temperature, while that of the fast mode is almost independent of temperature. The peak of the slow mode occurs at a slightly lower temperature than the transition temperature. (2) The dependence of the logarithm of the rate constants on the inverse of temperature (Arrhenius plot) is linear. Therefore, the plot of the free energy of activation vs temperature is linear. (3) The values of kinetic parameters obtained suggest that in the activated state the intramolecular interactions are half broken, while the state is close to the native state on the entropy axis. (4) The amino acid substitution, which is modeled as having slightly unfavorable short-range interactions, causes the substituted ones to be slightly unstable. Moreover, it causes the folding transition to slow. From the analysis of the way slowing down is observed in the two substituted models, we conclude that a structure, designed to model a β-sheet, is formed before it gets assembled with other structures, which are designed to model α-helices. The process of assembly occurs nearly at the activated state of the folding and unfolding transition. (5) It is suggested from this study that the maximum of folding rate constant in the Arrhenius plot that has been observed experimentally in real proteins is likely due to hydrophobic interactions.     

Folding thermodynamics of model four-strand antiparallel beta-sheet proteins.

The thermodynamic properties for three different types of off-lattice four-strand antiparallel beta-strand protein models interacting via a hybrid Go-type potential have been investigated. Discontinuous molecular dynamic simulations have been performed for different sizes of the bias gap g, an artificial measure of a model protein's preference for its native state. The thermodynamic transition temperatures are obtained by calculating the squared radius of gyration R(g)(2), the root-mean-squared pair separation fluctuation Delta(B), the specific heat C(v), the internal energy of the system E, and the Lindemann disorder parameter Delta(L). Despite these models' simplicity, they exhibit a complex set of protein transitions, consistent with those observed in experimental studies on real proteins. Starting from high temperature, these transitions include a collapse transition, a disordered-to-ordered globule transition, a folding transition, and a liquid-to-solid transition. The high temperature transitions, i.e., the collapse transition and the disordered-to-ordered globule transition, exist for all three beta-strand proteins, although the native-state geometry of the three model proteins is different. However the low temperature transitions, i.e., the folding transition and the liquid-to-solid transition, strongly depend on the native-state geometry of the model proteins and the size of the bias gap.     

Purification of recombinant proteins by fusion with thermally-responsive polypeptides.

Elastin-like polypeptides (ELPs) undergo a reversible, inverse phase transition. Below their transition temperature (Tt), ELPs are soluble in water, but when the temperature is raised above Tt, phase transition occurs, leading to aggregation of the polypeptide. We demonstrate a method for purification of soluble fusion proteins incorporating an ELP tag. Advantages of this method, termed "inverse transition cycling," include technical simplicity, low cost, ease of scale-up, and capacity for multiplexing. More broadly, the ability to environmentally modulate the physicochemical properties of recombinant proteins by fusion with ELPs will allow diverse applications in bioseparation, immunoassays, biocatalysis, and drug delivery.     

Maximal stabilities of reversible two-state proteins

The hydrophobic effect is the major force driving protein folding. Around room temperature, small organic solutes and hydrophobic amino acids have low solubilities in water and the hydrophobic effect is the strongest. These facts suggest that globular proteins should be maximally stable around room temperature. While this fundamental paradigm has been expected, it has not actually been shown to hold. Toward this goal, we have collected and analyzed experimental thermodynamic data for 31 proteins that show reversible two-state folding <--> unfolding transitions at or near neutral pH. Twenty-six of these are unique, and 20 of the 26 are maximally stable around room temperature irrespective of their structural properties, the melting temperature, or the living temperatures of their source organisms. Their average temperature of maximal stability is 293 +/- 8 K (20 +/- 8 degrees C). These proteins differ in size, fold, and number of domains, hydrophobic folding units, and oligomeric states. They derive from the cold-loving psychrophiles, from mesophiles, and from thermophiles. Analysis of the single-domain proteins present in this set shows that the variations in their thermodynamic parameters are correlated in a way which may explain the adaptation of the proteins to the living temperatures of the organisms from which they derive. The average energetic contribution of the individual amino acids toward protein stability decreases with an increase in protein size, suggesting that there may be an upper limit for protein maximal thermodynamic stability. For the remaining proteins, deviation of the maximal stability temperatures from room temperature may be due to greater uncertainties in their heat capacity change (DeltaC(p)) values, a weaker hydrophobic effect, and/or a stronger electrostatic contribution.     

The molecular basis of cooperativity in protein folding. Thermodynamic dissection of interdomain interactions in phosphoglycerate kinase.

In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature [Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278]. The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry. The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition. On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function. Analysis of the data indicates that at low temperatures the two domains behave independently of each other. The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains. These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds. This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK. It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.     

Heat capacity of hydrogen-bonded networks: an alternative view of protein folding thermodynamics

Large changes in heat capacity (deltaCp) have long been regarded as the characteristic thermodynamic signature of hydrophobic interactions. However, similar effects arise quite generally in order-disorder transitions in homogeneous systems, particularly those comprising hydrogen-bonded networks, and this may have significance for our understanding of protein folding and other biomolecular processes. The positive deltaCp associated with unfolding of globular proteins in water, thought to be due to hydrophobic interactions, is also typical of the values found for the melting of crystalline solids, where the effect is greatest for the melting of polar compounds, including pure water. This suggests an alternative model of protein folding based on the thermodynamics of phase transitions in hydrogen-bonded networks. Folded proteins may be viewed as islands of cooperatively-ordered hydrogen-bonded structure, floating in an aqueous network of less-well-ordered H-bonds in which the degree of hydrogen bonding decreases with increasing temperature. The enthalpy of melting of the protein consequently increases with temperature. A simple algebraic model, based on the overall number of protein and solvent hydrogen bonds in folded and unfolded states, shows how deltaCp from this source could match the hydrophobic contribution. This confirms the growing view that the thermodynamics of protein folding, and other interactions in aqueous systems, are best described in terms of a mixture of polar and non-polar effects in which no one contribution is necessarily dominant.     

Hydrophobicity of amino acid residues: differential scanning calorimetry and synthesis of the aromatic analogues of the polypentapeptide of elastin.

Relative hydrophobicities of aromatic amino acid residues are investigated by using differential scanning calorimetry (DSC) on 10 synthetic copolypentapeptides of poly(VPGVG) of elastin. Utilizing the hydrophobic-driven process of the inverse temperature transition exhibited by these polypentapeptides in aqueous solution, the relative hydrophobicities of Phe, Trp, and Tyr residues are determined by the critical temperature and heat of the transition. The DSC data for the aromatic residue containing copolypentapeptide aqueous solution indicate that tryptophan is the most hydrophobic amino acid residue, phenylalanine the third most hydrophobic on basis of transition temperature and the second on basis of transition heat. For tyrosine, significant differences are observed between the phenolic and the phenoxide anionic states. At pH 7, where tyrosine is protonated, it is found to be the second most hydrophobic amino acid residue on the basis of the transition temperature, whereas on the basis of the heat of transition, it is less hydrophobic than both tryptophan and phenylalanine. Changing the pH from pH 7 to pH 12, for example, for poly[0.8(VPGVG), 0.2(VPGYG)] in aqueous solution shifts the transition temperature from 7 to 49 degrees C with a dramatically reduced heat. On the basis of both the transition temperature scale and the heat of transition, the hydroxylated tyrosine appears less hydrophobic than glycine.     

Differential stabilization of two hydrophobic cores in the transition state of the villin 14T folding reaction

We report the distribution of hydrophobic core contacts during the folding reaction transition state for villin 14T, a small 126-residue protein domain. The solution structure of villin 14T contains a central beta-sheet with two flanking hydrophobic cores; transition states for this protein topology have not been previously studied. Villin 14T has no disulfide bonds or cis-proline residues in its native state; it folds reversibly, and in an apparently two-state manner under some conditions. To map the hydrophobic core contacts in the transition state, 27 point mutations were generated at positions spread throughout the two hydrophobic cores. After each point mutation, comparison of the change in folding kinetics with the equilibrium destabilization indicates whether the site of mutation is stabilized in the transition state. The results show that the folding nucleus, or the sub-region with the strongest transition state contacts, is located in one of the two hydrophobic cores (the predominantly aliphatic core). The other hydrophobic core, which is mostly aromatic, makes much weaker contacts in the transition state. This work is the first transition state mapping for a protein with multiple major hydrophobic cores in a single folding unit; the hydrophobic cores cannot be separated into individual folding subdomains. The stabilization of only one hydrophobic core in the transition state illustrates that hydrophobic core formation is not intrinsically capable of nucleating folding, but must also involve the right specific interactions or topological factors in order to be kinetically important.     

Reversibility and hierarchy of thermal transition of hen egg-white lysozyme studied by small-angle x-ray scattering

To clarify mechanisms of folding and unfolding of proteins, many studies of thermal denaturation of proteins have been carried out at low protein concentrations because in many cases thermal denaturation accompanies a great tendency of aggregation. As small-angle x-ray scattering (SAXS) measurements are liable to use low-concentration solutions of proteins to avoid aggregation, SAXS has been regarded as very difficult to observe detailed features of thermal structural transitions such as intramolecular structural changes. By using synchrotron radiation SAXS, we have found that the presence of repulsive interparticle interaction between proteins can maintain solute particles separately to prevent further aggregation in thermal denaturation processes and that under such conditions the thermal structural transition of hen egg-white lysozyme (HEWL) holds high reversibility even at 5% w/v HEWL below pH approximately 5. Because of the use of the high concentration of the solutions, the scattering data has enough high-statistical accuracy to discuss the thermal structural transition depending on the structural hierarchy. Thus, the tertiary structural change of HEWL starts from mostly the onset temperature determined by the differential scanning calorimetry measurement, which accompanies a large heat absorption, whereas the intramolecular structural change, corresponding to the interdomain correlation and polypeptide chain arrangement, starts much prior to the above main transition. The present finding of the reversible thermal structural transitions at the high protein concentration is expected to enable us to analyze multiplicity of folding and unfolding processes of proteins in thermal structural transitions.     

Differential scanning calorimetry studies of the inverse temperature transition of the polypentapeptide of elastin and its analogues

Differential scanning calorimetry studies have been carried out on the sequential polypeptide of elastin, (L-Val1-L-Pro2-Gly3-L-Val4-Gly5)n, abbreviated as PPP, and its more hydrophobic analogues (L-Leu1-L-Pro2-Gly3-L-Val4-Gly5)n, referred to as Leu1-PPP, and (L-Ile1-L-Pro2-Gly3-L-Val4-Gly5)n, referred to as Ile1-PPP Consistent with inverse temperature transitions, the temperatures of the transitions for which maximum heat absorption occurs are inversely proportional to the hydrophobicities of the polypentapeptides (31 degrees C for PPP, 16 degrees C for Leu1-PPP, and 12 degrees C for Ile1-PPP), and the endothermic heats of the transitions are small and increase with increasing hydrophobicity, i.e., 1.2, 2.9, and 3.0 kcal/mol pentamer for PPP, Leu1-PPP, and Ile1-PPP, respectively. Previous physical characterizations of the polypentapeptides have demonstrated the occurrence of an inverse temperature transition since increase in order, as the temperature is raised above that of the transition, has been repeatedly observed using different physical characterizations. Furthermore, the studies demonstrated identical conformations for PPP and Il21-PPP above and below the transition. Both heats and temperatures of the transitions vary with hydrophobicity, but not in simple proportionality.     

Probing the Folding-Unfolding Transition of a Thermophilic Protein,MTH1880

The folding mechanism of typical proteins has been studied widely, while our understanding of the origin of the high stability of thermophilic proteins is still elusive. Of particular interest is how an atypical thermophilic protein with a novel fold maintains its structure and stability under extreme conditions. Folding-unfolding transitions of MTH1880, a thermophilic protein from Methanobacterium thermoautotrophicum, induced by heat, urea, and GdnHCl, were investigated using spectroscopic techniques including circular dichorism, fluorescence, NMR combined with molecular dynamics (MD) simulations. Our results suggest that MTH1880 undergoes a two-state N to D transition and it is extremely stable against temperature and denaturants. The reversibility of refolding was confirmed by spectroscopic methods and size exclusion chromatography. We found that the hyper-stability of the thermophilic MTH1880 protein originates from an extensive network of both electrostatic and hydrophobic interactions coordinated by the central β-sheet. Spectroscopic measurements, in combination with computational simulations, have helped to clarify the thermodynamic and structural basis for hyper-stability of the novel thermophilic protein MTH1880.     

Dynamic light-scattering study on changes in flexibility of filamentous bacteriophage Pf1 with temperature.

The temperature dependence of the flexibility of bacteriophage Pf1 was investigated by dynamic light scattering, and the following results were obtained: The gamma/K2 values measured at 1 degree-25 degrees C and at various K values were T/eta-scaled to 20 degrees C, where gamma is the first cumulant of the field correlation function of scattered light, K is the length of the scattering vector, T is the absolute temperature, and eta is the solvent viscosity at T. And it was found that the scaled gamma/K2 values at low K values were independent of temperature, whereas those at high K values increased sigmoidally and reversibly against temperature. This suggests that the virion is more flexible at temperatures above the transition temperature Tt. This characteristic temperature Tt depended on the pH of the suspension: Tt = 11 degrees C at pH 6.9 and Tt = 8 degrees C at pH 8.2.     

Sequence–function relationships in folding upon binding

Folding coupled to binding is ubiquitous in biology. Nevertheless, the relationship of sequence to function for protein segments that undergo coupled binding and folding remains to be determined. Specifically, it is not known if the well-established rules that govern protein folding and stability are relevant to ligand-linked folding transitions. Upon small ligand biotinoyl-5′-AMP (bio-5′-AMP) binding the Escherichia coli protein BirA undergoes a disorder-to-order transition that results in formation of a network of packed hydrophobic side chains. Ligand binding is also allosterically coupled to protein association, with bio-5′-AMP binding enhancing the dimerization free energy by −4.0 kcal/mol. Previous studies indicated that single alanine replacements in a three residue hydrophobic cluster that contributes to the larger network disrupt cluster formation, ligand binding, and allosteric activation of protein association. In this work, combined equilibrium and kinetic measurements of BirA variants with alanine substitutions in the entire hydrophobic network reveal large functional perturbations resulting from any single substitution and highly non-additive effects of multiple substitutions. These substitutions also disrupt ligand-linked folding. The combined results suggest that, analogous to protein folding, functional disorder-to-order linked to binding requires optimal packing of the relevant hydrophobic side chains that contribute to the transition. The potential for many combinations of residues to satisfy this requirement implies that, although functionally important, segments of homologous proteins that undergo folding linked to binding can exhibit sequence divergence.     

A thermodynamic and kinetic analysis of the folding pathway of an SH3 domain entropically stabilised by a redesigned hydrophobic core

The folding thermodynamics and kinetics of the alpha-spectrin SH3 domain with a redesigned hydrophobic core have been studied. The introduction of five replacements, A11V, V23L, M25V, V44I and V58L, resulted in an increase of 16% in the overall volume of the side-chains forming the hydrophobic core but caused no remarkable changes to the positions of the backbone atoms. Judging by the scanning calorimetry data, the increased stability of the folded structure of the new SH3-variant is caused by entropic factors, since the changes in heat capacity and enthalpy upon the unfolding of the wild-type and mutant proteins were identical at 298 K. It appears that the design process resulted in an increase in burying both the hydrophobic and hydrophilic surfaces, which resulted in a compensatory effect upon the changes in heat capacity and enthalpy. Kinetic analysis shows that both the folding and unfolding rate constants are higher for the new variant, suggesting that its transition state becomes more stable compared to the folded and unfolded states. The phi(double dagger-U) values found for a number of side-chains are slightly lower than those of the wild-type protein, indicating that although the transition state ensemble (TSE) did not change overall, it has moved towards a more denatured conformation, in accordance with Hammond's postulate. Thus, the acceleration of the folding-unfolding reactions is caused mainly by an improvement in the specific and/or non-specific hydrophobic interactions within the TSE rather than by changes in the contact order. Experimental evidence showing that the TSE changes globally according to its hydrophobic content suggests that hydrophobicity may modulate the kinetic behaviour and also the folding pathway of a protein.     

Optimization of hydrophobic domains in peptides that undergo transformation from alpha-helix to beta-fibril

Recent studies on peptide fibrillogenesis by the de novo method as well as amyloidogenic proteins including prion proteins and Alzheimer's beta-peptides have provided insights into the conformational changes, such as alpha-helix to beta-structure, involved in folding and misfolding processes. We have found that an exposed hydrophobic nucleation domain at N-terminal causes a structural transition of a peptide from alpha-helix to beta-fibril. It became clear that N-terminal acyl groups of particular lengths in a 2alpha-helix peptide caused the peptide to undergo an alpha-to-beta transition. The peptide with the octanoyl group (C8-2alpha) showed the highest rate of transformation. The study of the designed peptides revealed that these alpha-to-beta transitions were closely related to the initial alpha-helix conformation and its stability. Engineering peptides that undergo alpha-to-beta transitions are attractive not only to the study of pathogenic proteins such as prion proteins, but also to the control of self-assembly of peptides, which will lead to the development of peptidyl self-assembling materials.     

Comparison of the conformation and orientation of alamethicin and melittin in lipid membranes

The secondary structure of alamethicin in lipid membranes below and above the lipid phase transition temperature Tt is determined by Raman spectroscopy and circular dichroism (CD) measurements. In both cases structural data are obtained by fitting the experimental spectra by a superposition of the spectra of 15 reference proteins of known three-dimensional structure. According to the Raman experiments, in a lipid bilayer above Tt alamethicin is helical from residue 1 to 12, whereas below Tt the helix extends from residue 1 to 16. The remaining C-terminal part is nonhelical up to the end residue 20 both above and below Tt. A considerable lower helix content is derived from CD, namely, 38% and 46% above and below Tt, respectively, in agreement with several reported values for CD in the literature. It is shown that the commonly used set of CD spectra of water-soluble reference proteins is unsuitable to describe the CD spectra of alamethicin correctly. Therefore the secondary structure of alamethicin as derived from CD measurements is at the present state of analysis unreliable. In contrast to the case of alamethicin, the CD spectra of melittin in lipid membranes are correctly described by the reference protein spectra. The helix content of melittin is determined thereby to be 72% in lipid membranes above Tt and 75% below Tt. The data are in accord with a structure where the hydrophobic part of melittin adopts a bent helix as determined recently by Raman spectroscopy [Vogel, H., & J?hnig, F. (1986) Biophys. J. 50, 573]. The orientational order parameters of the helical parts of alamethicin and of melittin in a lipid membrane are deduced from the difference between a corresponding CD spectrum of a polypeptide in planar multibilayers and that in lipid vesicles. The presented method for determining helix order parameters is new and may be generally applicable to other membrane proteins. The orientation of the helical part of both polypeptides depends on the physical state of the lipid bilayer at maximal membrane hydration and in the ordered lipid state furthermore on the degree of membrane hydration. Under conditions where alamethicin and melittin are incorporated in an aggregated form in a fluid lipid membrane at maximal water content the helical segments are oriented preferentially parallel to the membrane normal. Cooling such lipid membranes to a temperature below Tt changes the orientation of the helical part of alamethicin as well as melittin toward the membrane plane.(ABSTRACT TRUNCATED AT 400 WORDS)