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A genetic algorithm (GA) has been developed for the superimposition of sets of flexible molecules. Molecules are represented by a chromosome that encodes angles of rotation about flexible bonds and mappings between hydrogen-bond donor proton, acceptor lone pair and ring centre features in pairs of molecules. The molecule with the smallest number of features in the data set is used as a template, onto which the remaining molecules are fitted with the objective of maximising structural equivalences. The fitness function of the GA is a weighted combination of: (i) the number and the similarity of the features that have been overlaid in this way; (ii) the volume integral of the overlay; and (iii) the van der Waals energy of the molecular conformations defined by the torsion angles encoded in the chromosomes. The algorithm has been applied to a number of pharmacophore elucidation problems, i.e., angiotensin II receptor antagonists, Leu-enkephalin and a hybrid morphine molecule, 5-HT1D agonists, benzodiazepine receptor ligands, 5-HT3 antagonists, dopamine D2 antagonists, dopamine reuptake blockers and FKBP12 ligands. The resulting pharmacophores are generated rapidly and are in good agreement with those derived from alternative means.

Previous CD measurements of changes in the conformation of beta-lactoglobulin at neutral pH as a function of temperature indicated the formation of a molten globule state above approx. 70 degrees C. New CD measurements are reported at temperatures up to 80 degrees C with an instrument on the Daresbury synchrotron radiation source which gives spectra of good signal-to-noise ratio down to 170 nm. IR spectra were recorded up to 94.8 degrees C with a ZnSe circle cell and a single simplified model of the substructure of the amide I' band was used to give the fractional contents of beta-sheet structure unambiguously and independently of the CD spectroscopy. The results of both techniques, however, were in agreement in showing a progressive loss of beta-sheet structure with increasing temperature, beginning below the denaturation temperature. Nevertheless, the CD spectroscopy showed a fairly abrupt loss of virtually all the helical conformation at approx. 65 degrees C. Comparison of the present results with other studies on the molten globule formed at acid pH in the lipocalin family suggests that above 65 degrees C a partly unfolded state is formed, possibly by destabilization of the intermolecular beta-strand I and the loss of the main helix, but it is not a classical molten globule transition.

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We have investigated the effect of placing phosphoserine at the N-cap, N1, N2, N3, and interior position in alanine-based alpha-helical peptides. Helix contents of each peptide were measured by CD spectroscopy and titrations performed to determine pK(a) values. Data were analyzed with modified Lifson-Roig theory to determine helix-coil parameters (n, n(1), n(2), n(3), and w) and free energy changes for phosphoserine at each helical position. Results are given for a -1 and -2 phosphoserine charge state. Results show that phosphoserine stabilizes at the N-terminal positions by as much as 2.3 kcal.mol(-1), while destabilizes in the helix interior by 1.2 kcal.mol(-1), relative to serine. The rank order of free energies relative to serine at each position is N2 > N3 > N1 > N-cap > interior. Moreover, -2 phosphoserine is the most preferred residue known at each of these N-terminal positions. Experimental pK(a) values for the -1 to -2 phosphoserine transition are in the order N2 < N-cap < N1 < N3 < interior. This order agrees well with electrostatics calculations carried out with phosphoserine at the N-terminal positions and interior positions. Combining these with calculations at the C3, C2, C1, and C-cap positions gives results for phosphoserine along the length of the helix. We see a transition from phosphoserine stabilization at the N-terminus to destabilization at the C-terminus and can explain this in terms of the balance of protein solvation, favorable interactions, and dehydration. These results give insight into the phosphorylatable control of biological systems through positive or negative changes in stability.

It has long been believed that nucleation of the ␣-helix is a very fast reaction, occurring in around 10 ؊7 s. We show here that helix nucleation, in fact, takes place on the millisecond time scale. The rate of ␣-helix nucleation in two polyalanine-based peptides and in lysine and glutamic acid homopolymers was measured directly by stopped-f low deep UV CD with synchrotron radiation as the light source. Synchrotron radiation CD gives far superior signal to noise than a conventional instrument. The 16-aa AK peptide folds with first-order kinetics and a rate constant of 15 s ؊1 at 0°C. The rate-determining step is presumably the initiation of a new helix, which occurs at least 10 5 times slower than expected. Helix folding occurs in at least two steps on the millisecond time scale for the longer peptides, with a transient overshoot of helix content significantly greater than at equilibrium, similar to that seen in the folding of several proteins. We suggest that the overshoot is caused by the formation of a single long helix followed by its breakage into the two or more helices present at equilibrium.If we are to clearly understand protein folding, it is essential to understand the folding of the major substructures, the ␣-helix and ␤-sheet. Relaxation times for the helix͞coil transition of Glu and Lys homopolymers previously have been measured by electric field jump (1, 2), temperature jump (3-5), and resonant ultrasound methods (6-8). Temperature jump, IR spectroscopy (9), and N-terminal reporter group fluorescence (10) also have been applied to measure the kinetics of unfolding of a 21-residue poly(Ala)-based helical peptide.There are two microscopic rates in helix folding (11). First, there is the fast propagation of an existing helix by the addition of a single residue to the end of a helix. The rate of initiation of a new helix, presumably by the formation of a single turn, stabilized by one i, iϩ4 hydrogen bond, will be slow because it requires the entropically unlikely event of the simultaneous restriction of three successive residues. Previous results have been used to derive a rate for extension of helices by a single turn of 1 ϫ 10 7 to 7 ϫ 10 10 ⅐s Ϫ1and to infer very fast initiation rates for the coil-to-helix transition.Here we directly measure the rate of nucleation of ␣-helices from the denatured state. Helix formation in AK16 (sequence Ac-YGAAKAAAAKAAAAKA-NH 2 ) was initiated by a 10-fold dilution from 5 M GuHCl and in AQ28 (sequence Ac-A(QAAAA) 5 QGY-NH 2 ) by a 20-fold dilution from 6 M GuHCl. We also studied poly(L-lysine) and poly(L-glutamic acid). The 3-kDa poly(Lys) sample consisted of polymers in the molecular mass range of 1.5 to 4.5 kDa; 5-kDa poly(Lys) ranged from 1.6 to 10 kDa; 4.4-kDa poly(Glu) ranged from 3 to 14 kDa; 7-kDa poly(Glu) ranged from 3 to 22 kDa; and 20-kDa poly(Glu) ranged from 6 to 40 kDa. These polypeptides form ␣-helices when neutral and random coil when charged. Initiation of helix folding therefore was performed by a pH jump, from 8.0 to 11.5 for poly(Lys) an...

Fluorescence resonance energy transfer (FRET) was used to reveal aspects of the mechanism of signal transduction by epidermal growth factor receptors (EGFR). The superpositions of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha) and an antibody fragment (29.1) to the carbohydrate extremity of the receptor's ectodomain as measured by FRET, show that 14% of EGFRs in A431 cells are oligomerized before growth factor binding. After binding growth factor and signaling, these oligomers dissociate before releasing growth factor. Time courses of the FRET-derived distances between constitutively oligomerized EGFRs during signal transduction show a transient structural change in the extracellular domain, which occurs simultaneously with the production of intracellular Ca2+ signals. The FRET measurements also show a slow increase in oligomerization of EGFR monomers after growth factor binding. The structural change found in the extracellular domain of oligomeric EGFRs is similar to that shown by others for EPO, Neu, Fas, and tumor necrosis factor receptors, and may therefore be a common property of the transduction of the receptor-mediated signals.