The effect of enhanced alpha-helicity on the activity of a winter flounder antifreeze polypeptide.

The antifreeze polypeptide (AFP) from the winter flounder displays partial alpha-helix formation at lower temperatures. To investigate the relationship between antifreeze activity and alpha-helical structure, we designed and then chemically synthesized an AFP analog with enhanced alpha-helicity, and compared its conformation and antifreeze properties with those of the native AFP. The synthetic analog was more helical than the native AFP; however, the antifreeze activity of both peptides were identical. The antifreeze activity of the peptides displayed a strong pH dependence, which paralleled pH-induced changes in helix content. At pH 8.5, the antifreeze activity of both peptides displayed identical concentration dependences. In addition to antifreeze activity measurements, the effects of the peptides on the rate of ice crystal growth were also measured. While both peptides affected the a- and c-axis growth rates of ice crystals, the highly helical analog was able to exert its effect on ice crystal growth rates at 7-8-fold lower concentrations than the native AFP. These data indicate that there is a direct but complex relationship between alpha-helicity and antifreeze activity.     

ESR determination of membrane order parameter in yeast sterol mutants.

ESR investigations designed to determine membrane order parameter in sterol mutants of Saccharomyces cerevisiae were conducted using the membrane probe, 5-doxyl stearic acid. These mutants are blocked in the ergosterol biosynthetic pathway and thus do not synthesize ergosterol, the end product sterol. They do not require exogenous ergosterol for growth and, therefore, incorporate ergosterol biosynthetic intermediates in their membrane. Increasing order parameter is reflective of an increase in membrane rigidity. Single mutants involving B-ring delta 8 leads to delta 7 isomerization (erg 2) and C-24 methylation (erg 6) showed greater membrane rigidity than wild-type during exponential growth. A double mutant containing both lesions (erg 6/2) showed an even greater degree of membrane rigidity. During stationary phase the order of decreasing membrane rigidity was erg 6 greater than erg 6/2 greater than erg 2 = wild-type. The increased membrane order parameter was attributed to the presence of substituted sterols rather than increased sterol content or altered fatty acid synthesis.     

Bioactive N-terminal undecapeptides derived from parathyroid hormone: the role of alpha-helicity.

The N-terminal 1-34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses in bone characteristic of the native intact PTH. Recent studies have demonstrated that N-terminal fragments presenting the principal activating domain such as PTH(1-11) and PTH(1-14) with helicity-enhancing substitutions yield potent analogues with PTH(1-34)-like activity. To further investigate the role of alpha-helicity on biological potency, we designed and synthesized by solid-phase methodology the following hPTH(1-11) analogues substituted at positions 1 and/or 3 by the sterically hindered and helix-promoting C(alpha)-tetrasubstituted alpha-amino acids alpha-amino isobutyric acid (Aib), 1-aminocyclopentane-1-carboxylic acid (Ac(5)c) and 1-aminocyclohexane-1-carboxylic acid (Ac(6)c): Ac(5)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (I); Aib-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (II); Ac(6)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (III); Aib-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (IV); Aib-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (V); S-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (VI), S-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (VII); Ac(5)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (VIII); Ac(6)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (IX); Ac(5)c-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (X); Ac(6)c-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (XI). All analogues were biologically evaluated and conformationally characterized in 2,2,2-trifluoroethanol (TFE) solution by circular dichroism (CD). Analogues I-V, which cover the full range of biological activity observed in the present study, were further conformationally characterized in detail by nuclear magnetic resonance (NMR) and computer simulations studies. The results of ligand-stimulated cAMP accumulation experiments indicated that analogues I and II are active, analogues III, VI and VII are very weakly active and analogues IV, V, VIII-XI are inactive. The most potent analogue, I exhibits biological activity 3500-fold higher than that of the native PTH(1-11) and only 15-fold weaker than that of the native sequence hPTH(1-34). Remarkably, the two most potent analogues, I and II, and the very weakly active analogues, VI and VII, exhibit similar helix contents. These results indicate that the presence of a stable N-terminal helical sequence is an important but not sufficient condition for biological activity.     

Lipid-peptide association and activation of lecithin:cholesterol acyltransferase. Effect of alpha-helicity

A series of apolipopeptides, in which single proline substitutions were made at various sites in the 20-residue sequence, have been synthesized and tested. These peptides have nearly the same hydrophobic content, but very different helical contents, in a structure-making solvent. The affinity of these peptides for phospholipids was evaluated on the basis of their intrinsic tryptophan fluorescence and equilibrium dialysis against model high density lipoproteins. Proline substitutions at one end of the peptide had little or no effect on the fluorescence, circular dichroism, affinity for model high density lipoproteins, or activation of human plasma lecithin:cholesterol acyltransferase. By contrast, there was a dramatic change in all of these variables as the site of substitution was moved progressively closer to the middle of the peptide. All of these data suggested that a helix breaker that is substituted at the midpoint of a helical surface-associating peptide will greatly reduce its affinity for phospholipid surfaces. These results demonstrate that helicity and hydrophobicity are independent determinants of the affinity of an apolipopeptide for a phospholipid surface.     

Peptide alpha-helicity in aqueous trifluoroethanol: correlations with predicted alpha-helicity and the secondary structure of the corresponding regions of bovine growth hormone

The relationship between trifluoroethanol (TFE) enhancement of peptide alpha-helicity and protein secondary structure has been studied for a series of 11 peptides which span the complete primary sequence of bovine growth hormone (bGH). Ten of these peptides become increasingly alpha-helical as the solution concentration of TFE is increased. The amount of alpha-helicity developed by these peptides plateaus above 10 mol % TFE and ranges from 0 to 71%. The increased alpha-helicity, as determined by CD, closely correlates with the amount of alpha-helix predicted for eight of the eleven peptides analyzed (r = 0.9). Therefore, for this group of peptides, it appears that this technique can be used as a measure of alpha-helical propensity. Inclusion of the remaining three peptides in this analysis significantly lowers the correlation (r = 0.6). The reduced correspondence between TFE-enhanced and predicted alpha-helicity in this latter subset of peptides may be due to their relatively high hydrophobicity. In addition, the relevance of TFE-enhanced peptide alpha-helicity and the secondary structure of the corresponding protein regions was explored. Although the three peptides which form the largest amount of alpha-helicity in the presence of 10 mol % TFE correspond to alpha-helical regions of the protein, the overall correlation is significantly lower than is observed for the TFE-enhanced and predicted alpha-helicity. These findings suggest that the propensity of specific amino acid sequences for alpha-helix formation influences the amount of alpha-helicity which forms in corresponding protein sequences, but that other factors can modify this structure.     

The direct determination of protein structure by NMR without assignment.

Assignment of the resonances in nuclear magnetic resonance spectra is considered a pre-requisite for the interpretation of spectra that yield structural information. The determination of the three-dimensional structure of a biological macromolecule may, however, be achieved directly without spectral assignment, using the same set of heteronuclear scalar and dipolar coupling experiments as normally used. A cross-peak in any of the spectra may be interpreted as a distance between atoms, yielding a set of distances between unassigned atoms that serves to define the tertiary structure of the molecule. The principle is illustrated using the 76 amino acid protein ubiquitin.     

The chronoamperometric determination of homogeneous small molecule-redox protein reaction rates.

An essentially new application of chronoamperometry is presented for the determination of homogeneous second-order rate constants for the reactions between small molecule reductants and redox proteins. The first part of the work is a comparison between stopped-flow kinetics and chronoamperometric kinetics for the reaction of ferrous-EDTA with horse cytochrome c. The reaction was demonstrated to be first order in both ferrous-EDTA and cytochrome c and the effect of ionic strength was also studied. All of the chronoamperometric results compared well with the stopped-flow work which had been done previously. Chronoamperometry was then used to study several other reactions which have not been previously examined, including the reaction of ferrous-diethylenetriamine pentaacetic acid with cytochrome c. The reaction was slower than the ferrous-EDTA reaction but was more sensitive to ionic strength because of the greater charge (?3) on the complex. The second study was the reaction of ferrous-EDTA with Rhodospirillum rubrum cytochrome c₂ as a function of ionic strength. This novel application of chronoamperometry to small molecule-redox protein reactions represent a new and relatively easy alternative to anaerobic stopped-flow kinetics.     

The progress of membrane protein structure determination

The rate of membrane protein (MP) structure determination has been examined for the 18-year period following the publication of the first high-resolution crystal structure. The growth is solidly exponential, but lags behind the rate for soluble proteins during the equivalent time period.     

The structure determination of the variable portion of the Bence-Jones protein Au.

The structure of a pi-type Bence-Jones protein variable fragment Au has been determined by molecular replacement methods using the known structure of an other Bence-Jones variable fragment Rei (Epp et al., Eur J. Biochem. 45, 513 (1974). The crystallographic R factor is 0.31 for about 4000 significantly measured reflections between 6.8 to 2.5 A. The Au protein forms a dimer across a crystallographic two fold axis. The spatial relationship of the two monomers, the conformation of the backbones and of the internal residues is extremely similar to that found in Rei.     

ESR imaging of myelin basic protein induced vesicle aggregation

Using a modulated magnetic field gradient technique, the conventional ESR spectrum of well-defined spatial sections and the one-dimensional-ESR image of the nitroxide centre line of spin-labeled stearic acid in phospholipid vesicles were recorded with a spatial resolution of 4.10(-5) m after pelleting the vesicles inside 1 mm (i.d.) sample capillaries in a slow centrifuge (2500 X g). The sedimentation characteristics of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol vesicles were quantitatively compared with particular reference to vesicle aggregation induced by myelin basic protein. Protein-induced changes in the effective molecular mass were determined from ESR images of sedimentation profiles. The present data lend further support to the notion that the primary target of myelin basic protein-lipid interaction is the acidic lipid pool of myelin.     

The determination of lipopolysaccharide admixtures in protein preparations

A new method for the determination of lipopolysaccharide (LPS) admixtures in protein solutions has been developed. The method includes the periodate oxidation of LPS, biotinylation with biotin hydraside, immobilization on a nitrocellulose membrane and the development of biotinylated LPS in the streptavidin--alkaline phosphatase system. Proteins are previously removed from the solution by treatment with hot phenol. Development with the use of 5-bromoinodyl phosphate and nitrotetrazolium blue makes it possible to detect about 30 pg of LPS immobilized on the nitrocellulose membrane.     

Biophysical approaches to membrane protein structure determination.

Recently, there have been several technical advances in the use of solution and solid-state NMR spectroscopy to determine the structures of membrane proteins. The structures of several isolated transmembrane (TM) helices and pairs of TM helices have been solved by solution NMR methods. Similarly, the complete folds of two TM beta-barrel proteins with molecular weights of 16 and 19 kDa have been determined by solution NMR in detergent micelles. Solution NMR has also provided a first glimpse at the dynamics of an integral membrane protein. Structures of individual TM helices have also been determined by solid-state NMR. A combination of NMR with site-directed spin-label electron paramagnetic resonance or Fourier transform IR spectroscopy allows one to assemble quite detailed protein structures in the membrane.     

NMR and ESR studies on human pregnancy zone protein. Comparison with human alpha 2-macroglobulin.

NMR and ESR spectroscopies have been used to examine the plasma protease inhibitor pregnancy zone protein (PZP) and its complex with chymotrypsin. The 1H NMR spectrum of PZP shows relatively few sharp resonances, which, by analogy with human alpha 2-macroglobulin, probably arise from the proteolytically sensitive bait region. Upon reaction with chymotrypsin to form a 1:1 protease.PZP tetramer complex, there is a large increase in the intensity of sharp resonances due to an increase in mobility of these residues. 35Cl NMR has been used to follow binding of zinc and manganese to apo-PZP. Zinc binding causes a linear broadening of the bulk Cl-, consistent with access of Cl- to PZP-bound zinc. Since zinc in the two highest affinity sites in human alpha 2-macroglobulin causes no broadening of Cl-, it is concluded that these zinc sites are absent from PZP. The mobility of chymotrypsin in the PZP.chymotrypsin complex was examined by covalently attaching a nitroxide spin label at the serine residue in the active site of the enzyme and examining the appearance of the ESR spectrum. The chymotrypsin is rigidly held by the PZP to which it is covalently bound. In an analogous experiment performed previously on alpha 2-macroglobulin, chymotrypsin, bound in the presence of methylamine and therefore largely noncovalently bound, was found to be free to rotate inside the cage formed by the protease inhibitor.     

The optimization of protein secondary structure determination with infrared and circular dichroism spectra.

We have used the circular dichroism and infrared spectra of a specially designed 50 protein database [Oberg, K.A., Ruysschaert, J.M. & Goormaghtigh, E. (2003) Protein Sci. 12, 2015-2031] in order to optimize the accuracy of spectroscopic protein secondary structure determination using multivariate statistical analysis methods. The results demonstrate that when the proteins are carefully selected for the diversity in their structure, no smaller subset of the database contains the necessary information to describe the entire set. One conclusion of the paper is therefore that large protein databases, observing stringent selection criteria, are necessary for the prediction of unknown proteins. A second important conclusion is that only the comparison of analyses run on circular dichroism and infrared spectra independently is able to identify failed solutions in the absence of known structure. Interestingly, it was also found in the course of this study that the amide II band has high information content and could be used alone for secondary structure prediction in place of amide I.     

Evidence for the alpha-helicity of class II MHC molecular binding sites for the superantigen, staphylococcal enterotoxin A.

Circular dichroism (CD) spectra of class II MHC peptides revealed the alpha-helical conformation of superantigen-binding peptides I-A beta b(60-90), I-A beta b(65-85), and I-A alpha b(51-80), but not the nonbinding peptide I-A beta b(80-100). These CD spectra provide biophysical evidence for the alpha-helicity of class II MHC molecular binding sites for the superantigen, staphylococcal enterotoxin A (SEA). Alanine-substituted analogs of the SEA binding-site peptide, I-A beta b(65-85), were used to implicate beta-chain residues 72 and 80 in class II MHC-SEA binding. The data support SEA binding away from the class II antigen binding cleft along the faces of the alpha-helices.