Linus Pauling and the planar peptide bond - ualberta.ca · nature structural biology • volume 8...
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history nature structural biology • volume 8 number 3 • march 2001 201 Every first year biochem- istry student learns about the planarity of the pep- tide bond. Planarity, the result of ∼40% N–C′ dou- ble bond character arising from two dominant reso- nance structures, allows for a great simplification in the understanding of protein structures. Of the three repeating protein backbone dihedral angles, only φ and ψ need to be considered when assign- ing secondary structure, because the peptide bond dihedral angle ω is gener- ally considered fixed at 180°. Linus Pauling’s pre- diction of the α-helix, one of the greatest achieve- ments in structural biolo- gy, was made by assuming (i) that the peptide bond is planar, (ii) that all amino acid residues are equivalent with respect to backbone conformations and (iii) that each amide proton is hydrogen bonded to an oxygen atom of another residue with an N–O distance of 2.72 Å (ref. 1). Similar criteria allowed Pauling to predict several other protein secondary structural elements including the γ-helix and parallel and antiparallel β-sheets, accomplishments that resulted in his 1954 Nobel Prize in Chemistry. In 1968, Ramachandran recognized the need for nonplanar peptide bonds in cyclic peptides 2 . In later work, the same group argued that discrepancies in calcu- lated values of 3 J HN–Hα scalar coupling constants in peptides with bulky side chains showed the need to allow for non- planar peptide bonds in both cyclic and linear peptides 3 . They also noted that most of calculations up to that time used “the standard, completely planar, trans peptide unit of dimensions given by Pauling and Corey 4 as early as 1951” 3 . Much later and with relatively high level Hartree-Fock extended basis set ab initio calculations, Pople’s group showed that the peptide bond in the gas phase has sig- nificant flexibility and rotates as much as 40° with little energetic cost 5 , a result subsequently verified by Edison and coworkers 6 . Simple geometric drawings will show that deviations of this size result from relatively small changes in interatomic distances. In a statistical survey of peptide and protein databases, Thornton’s group con- vincingly showed that experimentally derived peptide and protein structures have significant deviations from planar peptide bonds 7 . Using data from 492 pep- tide bonds in the Cambridge Structural Database and 45,851 trans peptide bonds from 187 different protein structures in the Brookhaven Protein Data Bank (now the Research Collaboratory for Structural Bioinformatics Protein Data Bank), they were able to estimate the energies of pep- tide bond rotation using Maxwell- Boltzmann statistics (recreated in Fig. 1) 7 . Fig. 1 also provides updated values to Thornton’s survey that are in qualitative agreement with the results from 1996. Interestingly, in both the original study and updated values, there is a small but significant tendency toward angles <180°. Thus, both theory and experiment on both small peptides and proteins suggest that Linus Pauling’s famous planar pep- tide bond is overly simplistic. Could Linus Pauling, one of the world’s greatest chemists, have failed to understand that some flexibility is clearly a real (and quite likely important) property of the peptide bond? Several introductory and summa- ry statements made by Pauling show the impor- tance of peptide bond pla- narity: “The normal coplanarity of the atoms of [the peptide bond] is the result of resonance which gives rise to partial double bond character of the N–C′ peptide bond. Rotation about this bond is, in general, severely restricted.” (abstract of ref. 8); “The normal pla- narity of the amide group is established on both experimental and theoret- ical grounds as a sound structural principle. A structure in which the atoms of the amide group are not approximately coplanar should be regarded with scepticism until its rela- tively unstable configuration has been adequately confirmed.” (conclusion of ref. 8); “The N–C bond has about 40 per- cent double-bond character (bond length 1.32 Å). The group is planar, and it has been found to have the trans configura- tion in all substances studied except the cyclic peptides (diketopiperazine).” (page 498 of ref. 9). Pauling, like any great chemist, clearly saw the importance of generalization. He was a master at reducing complicated problems to their essence and stressing what he knew to be the most important points, which are emphasized as the major conclusions of his work. A more thorough reading, however, shows that he was keenly aware of the flexibility of the peptide bond: “About 40% double-bond character appears to be associated with the C′–N peptide bond. We may therefore estimate the strain energy involved in rotation around this bond. If the planes of the two ends of the amide group form a dihedral angle δ, and if A is the amide res- onance energy for the planar configura- tion, the strain energy may be taken equal to A sin 2 δ. A reasonable value for A is about 30 kcal/mole. From this we can calculate strain energies of about Linus Pauling and the planar peptide bond Arthur S. Edison Fig. 1 Peptide bond rotational energies and angular frequency distribution. The black and gray points are from ref. 7 and were derived from Maxwell-Boltzmann relations for proteins and peptides, respectively. The histogram represents the angular frequency distribution of 237,807 values of ω from coiled regions of 3,938 proteins in the current release of the protein data base (January, 2001) with resolu- tion ≤ 2.0 Å and R-factor ≤ 20%. The red points are energies derived from Maxwell- Boltzmann relations of the current values of ω, as described in ref. 7. The line is the function A sin 2 ω for A = 30 kcal mol –1 , as derived by Corey and Pauling in ref. 8. © 2001 Nature Publishing Group http://structbio.nature.com © 2001 Nature Publishing Group http://structbio.nature.com
Transcript of Linus Pauling and the planar peptide bond - ualberta.ca · nature structural biology • volume 8...
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nature structural biology • volume 8 number 3 • march 2001 201
Every first year biochem-istry student learns aboutthe planarity of the pep-tide bond. Planarity, theresult of ∼ 40% N–C′ dou-ble bond character arisingfrom two dominant reso-nance structures, allowsfor a great simplificationin the understanding ofprotein structures. Of thethree repeating proteinbackbone dihedral angles,only φ and ψ need to beconsidered when assign-ing secondary structure,because the peptide bonddihedral angle ω is gener-ally considered fixed at180°. Linus Pauling’s pre-diction of the α-helix, oneof the greatest achieve-ments in structural biolo-gy, was made by assuming(i) that the peptide bondis planar, (ii) that all amino acid residuesare equivalent with respect to backboneconformations and (iii) that each amideproton is hydrogen bonded to an oxygenatom of another residue with an N–Odistance of 2.72 Å (ref. 1). Similar criteriaallowed Pauling to predict several otherprotein secondary structural elementsincluding the γ-helix and parallel andantiparallel β-sheets, accomplishmentsthat resulted in his 1954 Nobel Prize inChemistry.
In 1968, Ramachandran recognized theneed for nonplanar peptide bonds incyclic peptides2. In later work, the samegroup argued that discrepancies in calcu-lated values of 3JHN–Hα scalar couplingconstants in peptides with bulky sidechains showed the need to allow for non-planar peptide bonds in both cyclic andlinear peptides3. They also noted thatmost of calculations up to that time used“the standard, completely planar, transpeptide unit of dimensions given byPauling and Corey4 as early as 1951”3.Much later and with relatively high levelHartree-Fock extended basis set ab initiocalculations, Pople’s group showed thatthe peptide bond in the gas phase has sig-nificant flexibility and rotates as much as40° with little energetic cost5, a resultsubsequently verified by Edison and
coworkers6. Simple geometric drawingswill show that deviations of this sizeresult from relatively small changes ininteratomic distances.
In a statistical survey of peptide andprotein databases, Thornton’s group con-vincingly showed that experimentallyderived peptide and protein structureshave significant deviations from planarpeptide bonds7. Using data from 492 pep-tide bonds in the Cambridge StructuralDatabase and 45,851 trans peptide bondsfrom 187 different protein structures inthe Brookhaven Protein Data Bank (nowthe Research Collaboratory for StructuralBioinformatics Protein Data Bank), theywere able to estimate the energies of pep-tide bond rotation using Maxwell-Boltzmann statistics (recreated in Fig. 1)7.Fig. 1 also provides updated values toThornton’s survey that are in qualitativeagreement with the results from 1996.Interestingly, in both the original studyand updated values, there is a small butsignificant tendency toward angles <180°.Thus, both theory and experiment onboth small peptides and proteins suggestthat Linus Pauling’s famous planar pep-tide bond is overly simplistic.
Could Linus Pauling, one of theworld’s greatest chemists, have failed tounderstand that some flexibility is clearly
a real (and quite likelyimportant) property ofthe peptide bond? Severalintroductory and summa-ry statements made byPauling show the impor-tance of peptide bond pla-narity: “The normalcoplanarity of the atomsof [the peptide bond] isthe result of resonancewhich gives rise to partialdouble bond character ofthe N–C′ peptide bond.Rotation about this bondis, in general, severelyrestricted.” (abstract ofref. 8); “The normal pla-narity of the amide groupis established on bothexperimental and theoret-ical grounds as a soundstructural principle. Astructure in which theatoms of the amide group
are not approximately coplanar shouldbe regarded with scepticism until its rela-tively unstable configuration has beenadequately confirmed.” (conclusion ofref. 8); “The N–C bond has about 40 per-cent double-bond character (bond length1.32 Å). The group is planar, and it hasbeen found to have the trans configura-tion in all substances studied except thecyclic peptides (diketopiperazine).” (page498 of ref. 9).
Pauling, like any great chemist, clearlysaw the importance of generalization. Hewas a master at reducing complicatedproblems to their essence and stressingwhat he knew to be the most importantpoints, which are emphasized as themajor conclusions of his work. A morethorough reading, however, shows that hewas keenly aware of the flexibility of thepeptide bond: “About 40% double-bondcharacter appears to be associated withthe C′–N peptide bond. We may thereforeestimate the strain energy involved inrotation around this bond. If the planes ofthe two ends of the amide group form adihedral angle δ, and if A is the amide res-onance energy for the planar configura-tion, the strain energy may be taken equalto A sin2δ. A reasonable value for A isabout 30 kcal/mole. From this we can calculate strain energies of about
Linus Pauling and the planar peptide bondArthur S. Edison
Fig. 1 Peptide bond rotational energies and angular frequency distribution. Theblack and gray points are from ref. 7 and were derived from Maxwell-Boltzmannrelations for proteins and peptides, respectively. The histogram represents theangular frequency distribution of 237,807 values of ω from coiled regions of 3,938proteins in the current release of the protein data base (January, 2001) with resolu-tion ≤ 2.0 Å and R-factor ≤ 20%. The red points are energies derived from Maxwell-Boltzmann relations of the current values of ω, as described in ref. 7. The line is thefunction A sin2ω for A = 30 kcal mol–1, as derived by Corey and Pauling in ref. 8.
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0.9 kcal/mole for 10 degree distortion ofthe amide group, 3.5 kcal/mole for 20degree distortion, and so on.” (page 14 ofref. 8).
A plot of Corey and Pauling’s 1953energy function for rotation of the pep-tide bond is shown alongside the 1996and most recent estimates based upon thestatistical survey of the peptide and pro-tein data bases. Nearly 50 years ago andwithout the benefit of a single high reso-lution protein structure or supercomput-er, Pauling got it right.
Tension can be a killer. This is especiallytrue when pressure builds up in single cellorganisms such as bacteria. To deal withrapid changes in osmotic pressure, bacte-ria have evolved safety valves in the form ofmechanosensitive channels. In manyorganisms such channels convert mechan-ical strain induced by sound, touch, gravi-ty or pressure into an electrochemicalresponse. When tension within the cellincreases, these channels open to releasepressure and osmolytes quickly and keepthe bacterium alive. While this channel is alifesaver in times of stress, at other times itneeds to be completely closed to maintainthe electrical integrity of the membrane. Aleaky channel would eventually kill thebacterium.
To gain an understanding of the gatingmechanism of such mechanosensitivechannels, Sukharev and coworkers (Nature409, 720–724 (2001)) developed a numberof structural models based on the previous-ly solved crystal structure of themechanosensitive channel of large conduc-tance, MscL, from Mycobacterium tubercu-losis (the closed structure was solved to 3.5 Å resolution) and previous patch clampexperiments on the Escherichia coli MscLchannel.
Features of the crystal structure and theenergetic parameters of the bacterial chan-nel were incorporated into a series of mole-cular models. The crystal structure showedthat the channel is composed of five identi-cal subunits (each one is a different color).Within each subunit, helical segments M1,M2 and S3 (shown as cylinders) wereobserved. The structure suggested that theM1 helices formed the gate.
Thermodynamic analyses of the MscL
Leak proof channel
channel predicted that the channel opensthrough pre-expanded intermediate states.The closed conformation (left) expands totwo-thirds of its open size to form the so-called closed-expanded conformation(middle) before it actually opens (right).Channel opening could occur by tilting ofthe M1 helices away from the center of thepore like the iris of an eye. Moreover, theability of the channel to go from theclosed-expanded conformation to theopen conformation without a substantialchange in the overall size of the channelsuggested the presence of a second gate.Given that S2 is poorly conserved and S3 isdispensable, the best candidate for a sec-ond gate was S1, which was disordered inthe crystal structure. Sukharev andcoworkers modeled the S1 segments asshort helices that block the pore when thechannel is in the closed conformation.
To test their model for the role of S1, theysubstituted cysteines for specific S1 residues
and asked whether these residues wereclose together in neighboring subunits inthe closed conformation and whethercrosslinking of S1 prevented channel open-ing. They found that S1 segments form abundle when the channel is closed andcrosslinking between S1 segments preventsopening. They also found that S1 segmentscrosslink with an outer helix (M2) whenthe channel is open, and this impedeschannel closing.
Thus, the MscL channel appears to havetwo gates in series to ensure that there is noleakage when the channel is closed. Thecytoplasmic (S1) gate ‘feels’ the tensiononly when the barrel is critically distortedand the flexible linkers connecting S1 toM1 are in the extended conformation. Inthis way, these channels serve as ‘emergencyrelease valves’ when the going gets toughand remain absolutely closed when life isstress free.
Boyana Konforti
AcknowledgmentI thank H. Weissig of the San Diego SupercomputerCenter and Protein Data Bank for providing thedihedral angles used in Fig. 1. D. Purich, R. McKenna, M. Agbandje-McKenna, and members of mylaboratory provided helpful and stimulatingdiscussions.
Arthur S. Edison is in the Department ofBiochemistry & Molecular Biology, Universityof Florida, Box 100245, Gainesville, Florida32610-0245. email: [email protected]
Received 7 December, 2000; accepted 31January, 2001.
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1. Pauling, L., Corey, R.B., and Branson, H.R. Proc. Natl.Acad. Sci. USA 37, 205–211 (1951).
2. Ramachandran, G.N. Biopolymers 6, 1494–1496 (1968).3. Ramachandran, G.N., Lakshminarayanan, A.V., and
Kolaskar, A.S. Biochim. Biophys. Acta 303, 8–13 (1973).4. Pauling, L., and Corey, R.B. Proc. Natl. Acad. Sci. USA
37, 235–240 (1951).5. Head-Gordon, T., Head-Gordon, M., Frisch, M. J.,
Brooks III, C.L., and Pople, J.A. J. Am. Chem. Soc. 113,5989–5997 (1991).
6. Edison, A.S., Markley, J.L., and Weinhold, F. J. Biomol.NMR, 4, 519–542 (1994).
7. MacArthur, M.W. and Thornton, J.M. J. Mol. Biol. 264,1180–1195 (1996).
8. Corey, R.B. and Pauling, L. Proc. Roy. Soc. B 141, 10–20(1953).
9. Pauling, L. The Nature of the Chemical Bond. (CornellUniversity Press, Ithaca, New York; 1960).
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