Pair Of Rotamers Research Articles

De novo prediction of explicit water molecule positions by a novel algorithm within the protein design software MUMBO

By mediating interatomic interactions, water molecules play a major role in protein–protein, protein–DNA and protein–ligand interfaces, significantly affecting affinity and specificity. This notwithstanding, explicit water molecules are usually not considered in protein design software because of high computational costs. To challenge this situation, we analyzed the binding characteristics of 60,000 waters from high resolution crystal structures and used the observed parameters to implement the prediction of water molecules in the protein design and side chain-packing software MUMBO. To reduce the complexity of the problem, we incorporated water molecules through the solvation of rotamer pairs instead of relying on solvated rotamer libraries. Our validation demonstrates the potential of our algorithm by achieving recovery rates of 67% for bridging water molecules and up to 86% for fully coordinated waters. The efficacy of our algorithm is highlighted further by the prediction of 3 different proteinligand complexes. Here, 91% of water-mediated interactions between protein and ligand are correctly predicted. These results suggest that the new algorithm could prove highly beneficial for structure-based protein design, particularly for the optimization of ligand-binding pockets or protein–protein interfaces.

Conformation Controls Mobility: 2H-Tetranaphthylporphyrins on Cu(111).

The adsorption behavior and the mobility of 2H‐Tetranaphthylporphyrin (2HTNP) on Cu(111) was investigated by scanning tunneling microscopy (STM) at room temperature (RT). The molecules adsorb, like the structurally related 2HTPP, in the “inverted” structure with the naphthyl plane restricted to an orientation parallel to the Cu surface. The orientation of the four naphthyl groups yields altogether 16 possible conformations. Due to the existence of rotamer pairs, 10 different appearances are expected on the surface, and all of them are identified by STM at RT. Most interestingly, the orientation of the naphthyl groups significantly influences the diffusion behavior of the molecules on Cu(111). We identify three different groups of conformers, which are either immobile, medium or fast diffusing at RT. The mobility seems to decrease with increasing size of the footprint of the conformers on the surface.

Helvamide, a new inhibitor of sterol O-acyltransferase produced by the fungus Aspergillus nidulans BF-0142.

A new piperazine derivative designated helvamide was isolated as a pair of rotamers (1 and 2) from the culture broth of the fungus Aspergillus nidulans BF-0142 along with a known helvafuranone (3). The structures of 1 and 2 were elucidated based on spectroscopic analyses by the interpretation of one-dimensional and two-dimensional nuclear magnetic resonance data, ROESY (rotational Overhauser effect spectroscopy) correlations, and a chemical method. Helvamide existed as a rotameric mixture (1 and 2) in dimethyl sulfoxide. Helvamide inhibited sterol O-acyltransferases 1 and 2 (SOAT1 and SOAT2) in enzyme-based and cell-based assays using SOAT1-expressing and SOAT2-expressing Chinese hamster ovary (CHO) cells.

Influence of Glu/Arg, Asp/Arg, and Glu/Lys Salt Bridges on α-Helical Stability and Folding Kinetics

Using a combination of ultraviolet circular dichroism, temperature-jump transient-infrared spectroscopy, and molecular dynamics simulations, we investigate the effect of salt bridges between different types of charged amino-acid residue pairs on α-helix folding. We determine the stability and the folding and unfolding rates of 12 alanine-based α-helical peptides, each of which has a nearly identical composition containing three pairs of positively and negatively charged residues (either Glu−/Arg+, Asp−/Arg+, or Glu−/Lys+). Within each set of peptides, the distance and order of the oppositely charged residues in the peptide sequence differ, such that they have different capabilities of forming salt bridges. Our results indicate that stabilizing salt bridges (in which the interacting residues are spaced and ordered such that they favor helix formation) speed up α-helix formation by up to 50% and slow down the unfolding of the α-helix, whereas salt bridges with an unfavorable geometry have the opposite effect. Comparing the peptides with different types of charge pairs, we observe that salt bridges between side chains of Glu− and Arg+ are most favorable for the speed of folding, probably because of the larger conformational space of the salt-bridging Glu−/Arg+ rotamer pairs compared to Asp−/Arg+ and Glu−/Lys+. We speculate that the observed impact of salt bridges on the folding kinetics might explain why some proteins contain salt bridges that do not stabilize the final, folded conformation.

Flavonol Glycosides with α‐Glucosidase Inhibitory Activities and New Flavone C‐Diosides from the Leaves of Machilus konishii

AbstractSeventeen flavonoids, five of which are flavone C‐diosides, 1–5, were isolated from the BuOH‐ and AcOEt‐soluble fractions of the leaf extract of Machilus konishii. Among 1–5, apigenin 6‐C‐β‐D‐xylopyranosyl‐2″‐O‐β‐D‐glucopyranoside (2), apigenin 8‐C‐α‐L‐arabinopyranosyl‐2″‐O‐β‐D‐glucopyranoside (4), and apigenin 8‐C‐β‐D‐xylopyranosyl‐2″‐O‐β‐D‐glucopyranoside (5) are new. Both 4 and 5 are present as rotamer pairs. The structures of the new compounds were elucidated on the basis of NMR‐spectroscopic analyses and MS data. In addition, the 1H‐ and 13C‐NMR data of apigenin 6‐C‐α‐L‐arabinopyranosyl‐2″‐O‐β‐D‐glucopyranoside (3) were assigned for the first time. The isolated compounds were assayed against α‐glucosidase (type IV from Bacillus stearothermophilus). Kaempferol 3‐O‐(2‐β‐D‐apiofuranosyl)‐α‐L‐rhamnopyranoside (12) was found to possess the best inhibitory activity with an IC50 value of 29.3 μM.

Biological and Structural Characterization of Rotamers of C-C Chemokine Receptor Type 5 (CCR5) Inhibitor GSK214096.

We recently reported the discovery of preclinical CCR5 inhibitor GSK214096, 1 (J. Med. Chem. 2011, 54, 756). Detailed characterization of 1 revealed that it exists as a mixture of four separable atropisomers A-D. The two slow-interconverting pairs of rotamers A + B and C + D were separated and further characterized. HIV and CCR5-mediated chemotaxis data strongly suggest that the antiviral potency of 1 is due to rotamers A + B and not C + D. Furthermore, integrated UV, vibrational circular dichroism VCD and computational approach allowed to determine the M chirality in C + D (and P chirality in A + B). These findings imply additional avenues to be pursued toward new CCR5 antagonists.

Biomolecular Structure Refinement & Prediction using Dead-End Elimination with a Polarizable Force Field

Global optimization of protein side-chains using discrete rotamer libraries is a challenging problem due to the large number of permutations that exist. For example, a small protein with 100 amino acid residues, each with 3 energetically favorable conformations, gives 3100 (or 1047) permutations to be searched. A systematic search over all permutations is computationally intractable, even in this simple case. To reduce the search space, rigorous inequalities have been described that eliminate high-energy rotamers, rotamer pairs, and so on (i.e. dead-end elimination), based on the assumption of a pairwise decomposable energy function. However, important biomolecular driving forces, including the hydrophobic effect and electronic polarization, are fundamentally many-body in nature. Neglect of higher order many-body forces has unduly limited the accuracy protein side-chain optimization and its applications to protein structure refinement, protein design and drug discovery. Here we present new rotamer elimination criteria that make possible the provable global optimization of protein side-chains using a polarizable (many-body) force field. Our many-body elimination criteria are only modestly more complex than the earlier pairwise versions when truncated at 3- or 4-body interactions. This opens the door to the rigorous use of not only polarizable force fields, which are now widely available for protein simulations, but also quantum mechanical potentials and/or implicit solvents such as Poisson-Boltzmann. Application to the protein “Proliferating Cell Nuclear Antigen” (PCNA) will be presented, which is a eukaryotic sliding clamp involved in numerous DNA processing pathways.

Polycyclic Hybrid Isoprenoids from a Reed Rhizosphere Soil Derived Streptomyces sp. CHQ-64

Two new hybrid isoprenoids, named indotertine B (2) and drimentine H (3), along with the known analogue drimentine C (4), were isolated from the reed rhizosphere soil derived actinomycete Streptomyces sp. CHQ-64. The structures of these compounds, including absolute configurations, were elucidated by extensive NMR, MS, and CD analyses. Indotertine B (2) exists as a pair of rotamers about the N-C(O) bond with a 2:1 ratio and displays activities against HCT-8 and A549 tumor cell lines with IC50 values of 6.96 and 4.88 μM, respectively.

Crucial Dependence of Chemiluminescence Efficiency on the Syn/Anti Conformation for Intramolecular Charge-Transfer-Induced Decomposition of Bicyclic Dioxetanes Bearing an Oxidoaryl Group

Thermally stable rotamers of bicyclic dioxetanes bearing 6-hydroxynaphthalen-1-yl (anti-5a and syn-5a), 3-hydroxynaphthalen-1-yl (anti-5b and syn-5b), and 5-hydroxy-2-methylphenyl groups (anti-5c and syn-5c) were synthesized. These dioxetanes underwent TBAF (tetrabutylammonium fluoride)-induced decomposition accompanied by the emission of light in DMSO and in acetonitrile at 25 °C. For all three pairs of rotamers, the chemiluminescence efficiency Φ(CL) for anti-5 was 8-19 times higher than that for syn-5, and the rate of CTID (charge-transfer-induced decomposition) for anti-5 was faster than that for syn-5. The chemiluminescence spectra of the rotamers for 5a and 5c, respectively, were different. This discrepancy in the chemiluminescence spectra between rotamers can presumably be attributed to the difference in the structures of de novo keto imide anti-14 and syn-14 in an excited state, which inherit the structures of the corresponding intermediary anionic dioxetanes anti-13 and syn-13. The important difference in chemiluminescence efficiency between anti-5 and syn-5 is discussed from the viewpoint of a chemiexcitation mechanism for CTID of oxidophenyl-substituted dioxetane.

C [sbnd]N bond rotation and E– Z isomerism in some N-benzyl- N-methylcarbamoyl chlorides: A DFT study

The current report presents the first theoretical study of the restricted C N bond rotation in carbamoyl chlorides. Several N-benzyl- N-methylcarbamoyl chlorides were investigated, with varying pattern of substitution in the aromatic ring. Optimizations and frequency calculations were conducted employing DFT at the B3LYP/6-31+G(d) level of theory. Each of the studied structures exhibits a pair of rotamers (s- Z and s- E), generated upon rotation around the C( O) N bond. The s- E isomer is the global minimum in every case, but the preference for it is usually less than 1 kcal/mol. Two possible transition state structures were identified for the rotamer interconversion: TS syn and TS anti , in close analogy to other related compounds, such as amides and carbamates. In contrast to the two latter types, however, the preferred transition state in the case of carbamoyl chlorides is TS syn , which we attribute to a stabilizing gauche effect. The optimized minima structures of the studied carbamoyl chlorides were subjected to GIAO B3LYP/6-311+G(2d,p) calculations, and the resultant isotropic shifts were found to be in excellent agreement with available experimental values. This has allowed us to provide unambiguous NMR signal assignments for the s- E and s- Z isomers of the studied compounds.

Computing van der Waals energies in the context of the rotamer approximation

The rotamer approximation states that protein side-chain conformations can be described well using a finite set of rotational isomers. This approximation is often applied in the context of computational protein design and structure prediction to reduce the complexity of structural sampling. It is an effective way of reducing the structure space to the most relevant conformations. However, the appropriateness of rotamers for sampling structure space does not imply that a rotamer-based energy landscape preserves any of the properties of the true continuous energy landscape. Specifically, because the energy of a van der Waals interaction can be very sensitive to small changes in atomic separation, meaningful van der Waals energies are particularly difficult to calculate from rotamer-based structures. This presents a problem for computational protein design, where the total energy of a given structure is often represented as a sum of precalculated rigid rotamer self and pair contributions. A common way of addressing this issue is to modify the van der Waals function to reduce its sensitivity to atomic position, but excessive modification may result in a strongly nonphysical potential. Although many different van der Waals modifications have been used in protein design, little is known about which performs best, and why. In this paper, we study 10 ways of computing van der Waals energies under the rotamer approximation, representing four general classes, and compare their performance using a variety of metrics relevant to protein design and native-sequence repacking calculations. Scaling van der Waals radii by anywhere from 85 to 95% gives the best performance. Linearizing and capping the repulsive portion of the potential can give additional improvement, which comes primarily from getting rid of unrealistically large clash energies. On the other hand, continuously minimizing individual rotamer pairs prior to evaluating their interaction works acceptably in native-sequence repacking, but fails in protein design. Additionally, we show that the problem of predicting relevant van der Waals energies from rotamer-based structures is strongly nonpairwise decomposable and hence further modifications of the potential are unlikely to give significant improvement.

Electron Transfer Prompted Ejection of a Tightly Bound K+ from the Ethereal Cavity of a Hexaarylbenzene-Based Receptor

[structure: see text]. Synthesis of a pair of rotamers (9u/9s) of a hexaarylbenzene derivative containing six (cofacially arranged) electroactive 2,5-dimethoxytolyl groups is described. The toroidal electronic stabilization due to the circular arrangement of aryl groups in 9u/9s leads to the observation of multiple (reversible) oxidation waves and lowering of their E(ox1) by approximately 250 mV relative to model compounds. The binding of K+ to symmetrical rotamer 9s was monitored by an electrochemical method and further confirmed by X-ray crystallography.

Dimeric anthocyanins from strawberry ( Fragaria ananassa) consisting of pelargonidin 3-glucoside covalently linked to four flavan-3-ols

Flavanol–anthocyanin complexes were isolated by successive use of Amberlite XAD-7 chromatography, Sephadex LH-20 gel filtration and preparative HPLC from acidified, methanolic extract of strawberries ( Fragaria ananassa Dutch.). These purple minor pigments were characterized by UV–Vis spectroscopy, 1D and 2D NMR techniques, and electrospray mass spectrometry to be catechin(4α→8)pelargonidin 3- O-β-glucopyranoside ( 1), epicatechin(4α→8)pelargonidin 3- O-β-glucopyranoside ( 2), afzelechin(4α→8)pelargonidin 3- O-β-glucopyranoside ( 3) and epiafzelechin(4α→8)pelargonidin 3- O-β-glucopyranoside ( 4). The stereochemistry at the 3- and 4-positions of the flavan-3-ol units was based on assumption of R-configuration at C-2. Each of the four pigments occurred in the NMR solvent as a pair of rotamers. Proved by cross-peaks in the 1H– 1H NOESY NMR spectra of 1, 2 and 4, the two conformations within each rotameric pair were in equilibrium with each other. Even though 1 and 2 are based on a different aglycone, their structures may be similar to tentatively identified pigments, which have been assumed to contribute to the colour of red wines.

Rate and equilibrium effects of tert-alkyl groups on rotamerization in 3,4-alkylenedioxy-, 3-alkoxy- and 3,4-dialkoxy-2-thienyl(di-tert-alkyl)methanols: an IR, NMR, kinetics and MM studyElectronic supplementary information (ESI) available: additional experimental data. See http://www.rsc.org/suppdata/p2/b2/b203714a/

Di-1-adamantyl and (1-adamantyl)(tert-butyl) analogues are compared with the previously studied 3- or 3- and 4-substituted 2-thienyl(di-tert-butyl)methanols. The equilibrium constants for syn–anti rotamerization ([syn]/[anti]) are slightly greater than for the smaller di-tert-butyl derivatives. In the intramolecularly hydrogen-bonded anti rotamers, neither the 1H NMR shift of the hydroxy proton nor the OH stretching frequency, both indicators of intramolecular hydrogen bond strength, is greatly affected by a change in the tert-alkyl group; these changes in the equilibrium constants must, therefore, be attributed to variations in steric effects. The rotation barriers for the compounds with the larger tert-alkyl groups are much enhanced and better differentiated. In all three sets the syn→anti and anti→syn rotation barriers are linearly correlated, and either can be correlated with the free energy difference. Nevertheless, the data do not meet the criteria for a classical Leffler–Grunwald-type rate-equilibrium relationship. Molecular mechanics calculations (MMFF94 force field) account fairly well for the variation in the free energy difference for rotamer pairs of the di-1-adamantyl, (1-adamantyl)(tert-butyl) and di-tert-butyl derivatives, but slightly overestimate the small differences in the equilibrium constants for the three series. The calculated rotation barriers for the di-tert-butyl compounds are about 7 kcal mol−1 higher than those observed. Those for 2-anisyl-, phenyl- and 2-tolyldialkylmethanols are increasingly overestimated as the rotation barrier rises, but a good correlation ranging over 20 kcal mol−1 is obtained; this correlation is not coincident with that for the thiophene derivatives.

Characterization of rotameric mixtures in o- and m-substituted benzaldehydes by matrix isolation IR spectroscopy.

The cis and trans conformers of benzaldehydes substituted in the o- or m-positions by Cl or CN can be differentiated by IR spectroscopy in Argon matrices. Partial photochemical rotamerization allows assignment of the IR bands and establishment of their relative intensities in pairs of rotamers, which permits, in turn, quantitation of equilibrium compositions prior to photolysis. The observed spectra and equilibrium compositions are in very good agreement with the predictions of vibrational spectra and free energy differences from B3LYP/6-31G calculations. The present work represents the first attempt to quantitate the small contributions that the cis rotamers make to the room-temperature equilibrium compositions of the two o-substituted benzaldehydes. The results for the m-substituted derivatives are compared to earlier estimates based on other methods.

Insertions of electrophiles into metal hydride bonds. Reactions of (C5Me5)Rh(PMe3)H2 with activated alkynes to produce η2-alkene complexes

The dihydride complex (C5Me5)Rh(PMe3)H2 (1) reacts with the electron deficient alkynes phenylacetylene (PhC≡CH), diphenylacetylene (PhC≡CPh), methyl propiolate (HC≡CE, E=CO2Me), dimethylacetylenedicarboxylate (EC≡CE, DMAD) and hexafluoro-2-butyne (F3CC≡CCF3) to produce in all cases η2-olefin complexes of the hydrogenated alkyne (2a–j). The complexes (C5Me5)Rh(PMe3)(η2-methacrylate) (2a, 2b), (C5Me5)Rh(PMe3)(η2-styrene) (2c,2d) and (C5Me5)Rh(PMe3)(η2-cis-stilbene) (2e, 2f) exist as pairs of rotamers that do not interconvert. The reaction of 1 with PhC≡CPh also produces (C5Me5)Rh(PMe3)(η2-trans-stilbene) (2g) at higher temperatures. Insertion of F3CC≡CCF3 into 1 occurs stereoselectively to yield (C5Me5)Rh(PMe3)(η2-trans-hexafluoro-2-butene) (2h). A mixture of cis- and trans- insertion products is observed in the reaction of 1 with EC≡CE to yield (C5Me5)Rh(PMe3)(η2-fumarate) (2i) and (C5Me5)Rh(PMe3)(η2-maleate) (2j) complexes. A crossover experiment using 1 and 1-d2 with DMAD shows only d0 and d2 η2-olefin products, demonstrating that both hydrogens come from the same molecule of 1. A second crossover experiment using (C5Me5)Rh(PMe3)(η2-fumarate)-d2 and free dimethylfumarate shows no evidence for incorporation of the free olefin. The vinyl hydride intermediates (C5Me5)Rh(PMe3)(CR=CHR)H are observed below −20°C for R=E and at room temperature for R=CF3. Cyclic voltammetry of the metal complex 1 and of the substituted alkynes shows irreversible behavior and half-wave redox potentials E1/2 are reported. CHCl3 quenching of the vinyl hydride products from the reaction of 1 with F3CC≡CCF3 yields crystals of the complex (C5Me5)Rh(PMe3)[trans-C(CF3)=CH(CF3)]Cl (5a) which was established as the trans complex by X-ray crystallography.

Rotamer-Specific Potentials of Mean Force for Residue Pair Interactions

We present an approach for the development of knowledge-based mean-force potentials for representing residue−residue interactions in proteins at the amino acid level of resolution which take into account the rotamer states of the residues. The rotamer-specific potentials which consist of an additional correction term to the current database-derived statistical potentials are expressed in terms of distance-dependent mean force potentials (MFP). To describe the relative geometry of two residues we used a set of six distances between different pairs of atoms. The distant-dependent MFPs are calculated from computer simulations of each pair of amino acids at the atomic level using the weighted-histogram analysis method. The umbrella sampling for each of 52003 different rotamer pairs was performed with a force-bias Monte Carlo algorithm using molecular mechanics potentials with statistically derived restraint terms. Our simulations show that the MFPs are very sensitive to the rotamer states of the amino acid residues. Preliminary tests of the potential's performance show that it can reproduce side-chain packing with reasonable accuracy.

Total, Asymmetric Synthesis of Methyl 3-Deoxy-3-(1′,2′,6′-trideoxy-2′,6′-imino-d-galactitol-1-yl)-α-d-manno-pyranoside (A C-Linked Imino Disaccharide): Conformational Analysis and Glycosidase Inhibition

The lithium enolate 16 derived in 5 steps from the Diels-Alder adduct of furan to 1-cyanovinyl (1S)-camphanate [(+)-12: a naked sugar of the first generation] was reacted with (+)(1R,4S,5S,6S)-6-endo-chroro-3-methylidene-5-exo-phenylseleno-7-oxabic yclo[2.2.1]. heptan-2-one [(+)-8] to give a unique product of Michael addition after acidic quenching. This adduct (-)-18 was transformed into branched long-chain carbohydrate derivatives such as benzyl 5-deoxy-5-[(5-O-acetyl-3-deoxy-2-O-methoxymethyl-D-manno-furanurono-6 , 1-lactone-3''-yl)methyl]- (tert-butyl)dimethylsilyl-2,3-di-O-methoxymethyl-alpha and beta-L-gulo-furanosideuronate [(-)-27]. Hydrogenolysis, followed by a Curtius rearrangement permitted to construct the corresponding branched 5-amino-5,6-dideoxydodecose derivative (-)-30 which was converted into 5-O-acetyl-3- [ 5'-N-(benzyloxy)carbonyl-2',3'-di-O-methoxymethyl-alpha and beta-D-manno-furanose]-2-O-methoxymethyl-D-manno-furanurono-6, 1-lactone [(-)-31] and then into methyl 3-deoxy-3-(1',2',6'-trideoxy-2',6'-imino-D-galactitol-1'-yl)-alpha-D-man no-pyranoside [(-)-1]. Its H-1 NMR spectrum measured at variable temperature demonstrated that (-)-1 exist as a double equilibrium involving two pairs of rotamers about the sigma C(1')-C(2') and sigma C(1')-C(3) bond [CH2 bridge linking beta-C(I) of the I,5-dideoxy-1,5-imino-D-lyxitol unit at C(3) of methyl alpha-D-manno-pyranoside]. Rotamers B' in which C(3) is gauche with respect to C(3') is preferred to conformers C' in which C(3) is anti with respect to C(3'). An equilibrium constant K (B' = C') congruent to 0.35 (25 degrees C) was evaluated. Rotamers B [C(2) gauche with respect to C(2')] and C [C(2) anti with respect to C(2')] are equally populated. Disaccharide mimetic (-)-1 is a moderate inhibitor of alpha-L-fucosidases from bovine liver (K-i = 100 mu M) and human placenta.

High-level electron correlation calculations on some tautomers of cytosine

Five isomers of cytosine, representing three tautomers and rotamers for two of them, have been investigated by ab initio techniques. Complete geometry optimizations were carried out at the Hartree-Fock (HF) and second-order perturbation theory, MBPT(2), levels. Our highest-level calculations used the coupled cluster method with single and double excitations to obtain energies at the MBPT(2) geometries. In the HF calculations basis sets up to 6-311G(2 d,2 p) were tested. The electron correlation calculations applied a DZP basis set. In the geometries, bond lengths reflect the varying formal bond orders of the tautomers, specifically the aromatic character of the pyrimidine ring in the amino-oxo tautomer. This latter form is found to be the most stable one in all calculations, in accordance with recent related literature results. It is found that its rotamer pair lies only about 0.8 kcal higher in energy. A qualitatively new result of the CCSD/DZP//MBPT(2)/DZP calculations is that the imino-oxo tautomer may be much more stable than thought previously, at only 0.2–0.3 kcal relative to the ground state. The order of energies in these high level calculations shows the pattern: amino-hydroxy < imino-oxo < amino-oxo.

Free energies of amino acid side-chain rotamers in α-helices, β-sheets and α-helix N-caps

Scales have previously been determined for the entropic cost of restricting amino acid side-chain rotations upon protein folding, giving the rule of thumb that the entropic cost of restricting a single side-chain bond is ≈0.5 kcal mol−1. However, this result does not consider the distinct preferences shown by amino acid side-chains for particular side-chain χ1 angles in the folded protein. For example, Glu in an α-helix has χ1 4% gauche− (g−), 39% trans (t) and 58% gauche+ (g+) showing that it is most favourable to restrict Glu χ1 as g+ in a helix while g− is least favoured. The change in side-chain conformational entropy is the same in both cases, but the free energy of each rotamer is different. Here, we determine the energies of every amino acid χ1 rotamer in α-helices, β-sheets and α-helix N-caps and each χ1χ2 rotamer pair in helices and sheets. The calculation uses observed rotamer distributions in secondary structure and the coil state, together with experimentally determined free energy changes for secondary structure formation. The results are sets of rotamer energies within a secondary structure that can be directly compared to each other. For example, we conclude that Tyr is the most stable residue in a β-sheet if only the trans rotamer is accessible; if only the gauche− conformation is available, Thr would be the most stabilising. Previously published scales of amino acid preferences for secondary structure are weighted averages of rotamer energies and therefore imply that Thr is the most stabilising substitution in a β-sheet in any side-chain conformation. Both side-chain conformational entropies and intrinsic secondary structure preferences are subsumed within our data; the results presented here should therefore be used in preference to both side-chain conformational entropies and intrinsic secondary structure preferences when the rotamer occupied in the folded state is known. The results may be useful in protein engineering, simulations of binding and folding, prediction of protein stability and peptide binding energies, and identification of incorrectly folded structures.