The hydration of amides in helices; a comprehensivepicture from molecular dynamics, IR, and NMR

SCOTT T.R. WALSH,1,3,4, RICHARD P. CHENG,1,3 WAYNE W. WRIGHT,1

DARWIN O.V. ALONSO2, VALERIE DAGGETT,2 JANE M. VANDERKOOI,1 AND

WILLIAM F. DEGRADO1

1Department of Biochemistry and Biophysics, University of Pennsylvania,Philadelphia, Pennsylvania 19104-6059, USA2Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195-7610, USA

(RECEIVED July 3, 2002; FINAL REVISION November 15, 2002; ACCEPTED November 15, 2002)

Abstract

We examined the hydration of amides of �3D, a simple, designed three-helix bundle protein. Moleculardynamics calculations show that the amide carbonyls on the surface of the protein tilt away from the helicalaxis to interact with solvent water, resulting in a lengthening of the hydrogen bonds on this face of the helix.Water molecules are bonded to these carbonyl groups with partial occupancy ( ∼ 50%–70%), and theirinteraction geometries show a large variation in their hydrogen bond lengths and angles on the nsec timescale. This heterogeneity is reflected in the carbonyl stretching vibration (amide I� band) of a group ofsurface Ala residues. The surface-exposed amides are broad, and shift to lower frequency (reflectingstrengthening of the hydrogen bonds) as the temperature is decreased. By contrast, the amide I� bands of theburied 13C-labeled Leu residues are significantly sharper and their frequencies are consistent with theformation of strong hydrogen bonds, independent of temperature. The rates of hydrogen-deuterium ex-change and the proton NMR chemical shifts of the helical amide groups also depend on environment. Thepartial occupancy of the hydration sites on the surface of helices suggests that the interaction is relativelyweak, on the order of thermal energy at room temperature. One unexpected feature that emerged from thedynamics calculations was that a Thr side chain subtly disrupted the helical geometry 4–7 residues N-terminal in sequence, which was reflected in the proton chemical shifts and the rates of amide protonexchange for several amides that engage in a mixed 310/�/�-helical conformation.

Keywords: Hydrogen bonding in helices; molecular dynamics; isotope-edited infrared spectroscopy;nuclear magnetic resonance; de novo protein design

The interaction of proteins with water plays a large andessential role in almost all aspects of protein folding andfunction (Mattos 2002). For example, when helices areformed during the folding of a protein, their amides arepartially to fully dehydrated; the degree of residual solventdepends on their location in the protein (Baker and Hubbard1984; Thanki et al. 1988). Carbonyl oxygen groups on thesurface of proteins often retain the ability to form a hydro-gen bond to water, while simultaneously forming the nor-mal helical hydrogen bond to an amide proton at positioni + 4 in the sequence (Baker and Hubbard 1984; Thanki etal. 1988). Such interactions have been proposed to be im-portant to the stability of the �-helix, as assessed from mo-lecular dynamics simulations (Daggett and Levitt 1992),

Reprint requests to: Valerie Daggett, Department of Medicinal Chem-istry, University of Washington, Seattle, WA 98195-7610, USA; e-mail:daggett@u.washington.edu; fax: (206) 685-3252; or Jane M. Vanderkooi,Department of Biochemistry and Biophysics, University of Pennsylvania,Philadelphia, PA 19104-6059, USA; e-mail: vanderko@mail.med.upenn.edu; fax: (215) 573-2042; or William F. DeGrado, Department ofBiochemistry and Biophysics, University of Pennsylvania, Philadelphia,PA 19104-6059, USA; e-mail: wdegrado@mail.med.upenn.edu; fax: (215)573-7229.

3These authors contributed equally to this work.4Present address: Department of Biochemistry and Molecular Biology,

University of Chicago, 920 E. 58th Street, Chicago, IL 60637-1474, USA.Abbreviations: NMR, nuclear magnetic resonance; IR, infrared; �,

chemical shift; HPLC, high performance liquid chromatography; nsec,nanosecond; MD, molecular dynamics.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0223003.

Protein Science (2003), 12:520–531. Published by Cold Spring Harbor Laboratory Press. Copyright © 2003 The Protein Society520

and in conjunction with measurements of the stability ofvariants of Ala-rich peptides that form monomeric helices(Luo and Baldwin 1999; Avbelj et al. 2000). However, thereare few methods available to directly assess the extent andstrength of such hydrogen bonds to water molecules withinthe helices of folded proteins. X-ray and neutron diffractionhave provided the primary methods to examine the presenceof water bound to specific sites within proteins (Savage andWlodawer 1986; Schoenborn et al. 1995). However, thedensity associated with water molecules is often not welldefined, because of disorder or partial occupancy. Also, thecrystal structures of proteins are generally solved near liquidnitrogen temperature, which has a dramatic effect on thehydration of the amides as demonstrated in this manuscript.NMR has also been used to explore waters of hydration(Otting et al. 1991; Belton 1994; Otting 1997; Halle andDenisov 2001; Mattos 2002), although only a subset of thecrystallographically defined water molecules are typicallyobserved. Here, we use temperature-dependent IR andNMR spectroscopy together with molecular dynamics simu-lations to examine the extent of solvation of specificallylabeled carbonyl groups within a small helical protein. Theresults advance our understanding of this problem whilesimultaneously providing simple methods to assess the de-gree of interaction between water and helical carbonylgroups.

In �-helices of proteins, the amide proton typically formsonly a single hydrogen bond (Baker and Hubbard 1984;Thanki et al. 1988). The larger size of the carbonyl oxygenand the presence of lone-pair electrons allow the formationof an additional hydrogen bond to water or other polar sidechains (often a Ser or Thr located at positions i + 4 or i + 3in the helix) (Baker and Hubbard 1984; Thanki et al. 1988).To accommodate this additional hydrogen bond, the amidecarbonyl groups on the solvent-accessible side of a helixtend to tilt away from the helical axis by about 20° (Blundellet al. 1983; Baker and Hubbard 1984; Chakrabarti et al.1986; Barlow and Thornton 1988; Thanki et al. 1988); this

tilt increases the length of the intrahelical hydrogen bondand decreases its linearity (Fig. 1). These geometric differ-ences between solvent-accessible and solvent-inaccessibleamide carbonyl groups (Chakrabarti et al. 1986; Olivella etal. 2002) have major affects on their spectroscopic proper-ties. For example, there is a general trend of proton chemi-cal shifts of the amide protons on the solvent-accessible sideof the helices tend to be shifted upfield (Goodman and Kim1991; Wishart et al. 1991; Milne et al. 1998; Cordier andGrzesiek 2002), as compared to the more strongly hydro-gen-bonded amide protons on the solvent-inaccessible sidesof helices. The location of the amides within helices alsoinfluences their rates of exchange with protons from solvent(Goodman and Kim 1991; Milne et al. 1998). Further, thesolvent-inaccessible amides have lower temperature coeffi-cients (��/�T) (Anderson et al. 1997; Baxter and William-son 1997), although the correlation is not as great as thatwith chemical shift and hydrogen-deuterium exchange.Therefore, in this manuscript, we focused on the former twomeasurements.

The degree of exposure of amides also has a major effecton the amide I� band, a mode that primarily reflects thecarbonyl stretch (the prime indicates that the amides aredeuterated, which is required to minimize the absorbance ofthe solvent). Recently, we have used isotopic labeling todetermine how solvent-accessibility and temperature affectthe amide I� mode, �c�o, in the �-helical coiled coil fromGCN4 (GCN4-P1�) (Manas et al. 2000). The 13C-labeledamide I� band occurred at lower energy for solvent exposed(1585 cm−1) versus buried positions (1606 cm−1). Thesedata agree with previous studies on unlabeled monomerichelices and coiled coils, which suggested that the solventaccessibility of a helical amide might decrease the fre-quency of �c�o. As the strength of the hydrogen bonds to acarbonyl group increases, �c�o generally decreases, in partreflecting a weakening of the C�O bond. Thus, the de-crease in the energy of the C�O stretch for exposed amidesin helices appears to be a result of their receiving an addi-

Figure 1. Exposed (left) and buried (right) amide in a helix of �3D. The schematics provide an idealized description of the geometriesof the two groups, as well as their expected spectroscopic properties.

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tional hydrogen bond from solvent, which more than com-pensates for the decrease in the hydrogen-bond strengthassociated with the intramolecular helical hydrogen bonds.The temperature dependence of the amide I� band also pro-vides important environmental information; in our previousstudy, �c�o of the solvent-exposed amides decreased mark-edly with lowering temperature, presumably reflecting anincrease in the strength of the intermolecular hydrogen bondto solvent. By contrast, the amide I� band of the buriedamides were nearly independent of temperature. Finally, the�c�o for the solvent-exposed amide showed a dramaticsharpening at lower T, possibly reflecting an increase in thehomogeneity and the occupancy of the hydrogen bonds tosolvent.

In this manuscript, we confirm and extend these findingsby examining a designed globular protein, �3D, which hasnumerous advantages compared to other natural or designedsystems. The globular structure of �3D is more typical ofsmall globular proteins than the elongated coiled coils thatwere studied previously. Further, the protein is stabilized bya relatively simple hydrophobic core, with no contributionsfrom long-range hydrogen bonds that might otherwise com-plicate the analysis. The solution structure of �3D has beendetermined (Walsh et al. 1999), and the dynamics of itsside-chain and main-chain atoms have been extensivelycharacterized by NMR (Walsh et al. 2001a), which indicatesthat the S2 order parameters of the main-chain amides aresimilar to those of natural globular proteins. Finally, there isextensive sequence homology between the three helices,which allows one to average the values of various spectro-scopic parameters. Thus, it should be possible to minimizethe contributions from variations in local conformation andchemical composition, allowing one to discern more generaltrends that are associated with solvent exposure of the he-lices.

Results and discussion

Design and preparation of 13C-labeled samples forIR spectroscopy

For IR spectroscopy, it is desirable to label a subset of theamide groups, allowing one to differentiate solvent-exposedfrom solvent-inaccessible carbonyls. Figure 2 illustrates thestructure of �3D, with the exposure of the carbonyl groupsof the Leu and Ala side chains coded according to the extentto which they form hydrogen bonds to solvent in the mo-lecular dynamics calculations described below. All but twoof the seven leucyl residues in �3D are essentially fullyburied from solvent. Conversely, 13 of the 15 Ala residuesare exposed to solvent. Thus, we prepared samples in whichthe amides of either the Ala or Leu were biosyntheticallylabeled at the carbonyl group with 13C. To minimize thecontribution from Ala60, which is buried in the core of theprotein, we used a previously well-characterized mutant of�3D, Ala60Ile (Walsh et al. 2001b). For simplicity, we willrefer to this derivative as �3D� in the sections below.

Molecular dynamics

The motion of �3D was simulated in a periodic box of water(∼ 3000 water molecules) using periodic boundary condi-tions to minimize edge effects. The simulation was per-formed at 300 K for 10 nsec. A number of parameters,including various torsions, angles, and bond lengths, weremonitored to determine which were most appropriate forcomparison with the spectroscopic observations. Ulti-mately, three parameters were found to correlate well withthe experimental observations: (1) dNH-O, the �-helical hy-drogen bond length (the distance between a carbonyl atposition i and an amide hydrogen at i + 4); (2) ∠ NH-O, the

Figure 2. Structure of �3D, with the percent exposure of the carbonyl oxygen atoms coded according to color. On the left are the Alaamides, while the Leu amides are at right.

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522 Protein Science, vol. 12

�-helical hydrogen bond angle (the angle formed by thecarbonyl oxygen at i, and the amide proton and nitrogen ati + 4); and (3) the percentage of structures in which a givencarbonyl is hydrogen-bonded to one or more solvent mol-ecules, nHOH-O, or the water occupancy about a given car-bonyl group.

These three parameters were measured for each helix in�3D, and are illustrated in Figure 3. The parameters gener-ally show a periodic distribution, with a repeat matchingthat of the �-helix. However, helix 1 deviates from thispattern, particularly with respect to the hydrogen bondlength and distance. A less substantial deviation from heli-cal periodicity is also apparent at approximately the sameposition in helix 2. Examination of the structures indicatedthat the perturbation of the helical periodicity in helix 1 wasa result of a persistent (98% occupancy) and strong (average

distance 1.63Å) hydrogen bond from the Thr16 side chain toa carbonyl group four residue N-terminal in sequence (Fig.4). This hydrogen bond caused a disruption of the helicalgeometry of residues 9–12, which show mixed 310/�-helicalcharacter. Two waters form hydrogen bonds with the car-bonyl groups of residues 8 and 9, which stabilize the 310–�-helical junction. Significantly, these sites on the proteinhave the highest water occupancies (Fig. 3). The structuralreorganization N-terminal to Thr16 can also be seen in the� and � angles of the amides (data not presented). Thorntonand coworkers (Blundell et al. 1983) have noted an anticor-relation between the � and � torsional angles at individualpositions in �-helices. Indeed, these correlated changesfrom the ideal helical values of � and � contribute to bend-ing of helices on the surface of proteins. A similar negativecorrelation (correlation coefficient −0.75) was observed for

Figure 3. The parameters nHOH-O, dNH-O, and ∠ NH-O as a function of the position in the sequence of �3D. The values are averages over 2–10 nsec involving40,000 structures.

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Figure 4. (A) An intramolecular hydrogen bond involving Thr16 disrupts the local helical geometry in helix 1 of �3D. (B) A ribbonrepresentation of �3D with the Thr16 side chain in stick representation; the core residues and Ala’s are shown in green and magenta,respectively. (C) Stereo-view of the intramolecular hydrogen bond involving Thr16 in helix 1; the side chain of the core residues andAla’s are shown in green and magenta, respectively. (D) Stereo-view of �3D with waters hydrogen-bonded to the backbone shown;side chains are omitted for clarity. The C� of the core residues and Ala’s are shown in green and magenta, respectively.

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the helical positions of �3D, except for residues 9–12, justN-terminal from Thr16 (data not shown).

Ser40 occurs at a position analogous to Thr16, and simi-larly engages in a hydrogen bond to a carbonyl at positioni-4 in sequence. However, the mean occupancy of this in-tramolecular hydrogen bond was 34% versus 98% forThr16. Blaber and coworkers (Blaber et al. 1994) haveshown that the �-methyl group of Thr specifically favorsthis side-chain to main-chain hydrogen bond by destabiliz-ing alternate conformations available to Ser, thus, the meanstructural perturbation associated with the side-chain tobackbone hydrogen bond at Ser40 appeared to be less se-vere.

The static and dynamic variations in dNH-O, ∠ NH-O, andnHOH-O at a given position of �3D will affect the IR andNMR spectra differently. The hydrogen bonds to water gen-erally show partial rather than full occupancy (Fig. 3), andthe geometry of the water-carbonyl hydrogen bonds at agiven site vary significantly with respect to time (notshown). Also, dNH-O and ∠ NH-O vary significantly for sol-vent-exposed residues, while they are essentially constantfor the most buried residues. Of particular relevance to theNMR measurements, all three parameters equilibrate rap-idly on the 1-nsec time scale. Thus, we would expect theamides to be rapidly exchanging with respect to the NMRmeasurements discussed below; thus, one should observesingle sharp peaks whose positions reflect the population ofthe various configurations. In contrast, the IR measurementsoccur on the time scale of a molecular vibration, such thatwe expect to see a broad spectral distribution that reflectsthe full ensemble of molecular configurations about eachamide bond. More specifically, the dynamics calculationspredict that, at room temperature, the amide I� band shouldbe significantly broader for the exposed carbonyl groups ascompared with the buried carbonyls.

Hydrogen bonds between the terminal guanidinium andammonium groups of Arg and Lys, respectively, have beenproposed to stabilize helices by interacting with the car-bonyl groups four residues N-terminal to these residues inthe sequence of �-helices (Vila et al. 2000, 2001; Garciaand Sanbonmatsu 2002). We therefore measured the fre-quency with which these residues of �3D interacted with theappropriate carbonyls in the helix (data not presented). Nosignificant hydrogen bonding was observed, indicating thatif this interaction actually stabilizes the structures of helices,it may be limited to the monomeric, Ala-rich helices inwhich it has been predicted to occur. This conclusion alsoagrees with surveys of protein structure, which failed toidentify this interaction as a feature that occurred frequentlyin �-helices (Baker and Hubbard 1984).

IR spectra of �3D�

The IR spectra of unlabeled �3D’ at temperatures rangingfrom 300 to 13 K are shown in Figure 5A. The bands in the

range from 1610 to 1660 cm−1 arise primarily from theamide I� band, while the band at ∼ 1670 cm−1 is from tri-fluoracetate, which is present as a counterion to the Lys andArg side chains following purification by reverse-phaseHPLC. At room temperature, the amide I� region is broaderthan observed for N-methylacetamide (Manas et al. 2000),suggesting the presence of more than one overlappingbands. Indeed, as the temperature is lowered, the broad peakin the �3D� spectrum splits into two distinct components,with maxima at ∼ 1630 and 1645 cm−1, respectively. Byanalogy to earlier studies (Manas et al. 2000), we assignthese peaks to exposed and buried helical amides, respec-tively (Fig. 3). The position of the lower-energy band of�3D�, which is associated with solvent-accessible amides,depends markedly on temperature, while the band associ-

Figure 5. (A) Temperature dependence of the IR spectra of unlabeled; (B)13C-Ala-labeled; and (C) 13C-Leu-labeled �3D�. Spectra were collectedfrom 290 K and 13 K (direction indicated by arrow) in D2O-glycerol-d8

(50:50 v/v) at pD 7.0.

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ated with solvent-inaccessible amides is largely independentof solvent.

Figure 5B illustrates that 13C-labeling of the Ala residueson the solvent-exposed face of the helices results in a newfeature in the amide I� region of the spectrum. As expectedfrom the molecular dynamics calculations, this peak is quitebroad at room temperature, but resolves at low temperaturesinto a well-resolved peak at ∼ 1590 cm−1. A second featureevident in the spectra is a diminution in the intensity of thepeak associated with the unlabeled exposed amides (at 1630cm−1 in the low-temperature spectra). Thus, the amide I�frequencies of the 13C-labeled and unlabeled Ala amides are1590 and 1630 cm−1, respectively. The isotope shift is ∼ 40cm−1, which is close to the value of 37 cm−1, expected foran unperturbed oscillator (Decatur and Antonic 1999).

Different behavior is observed when the Leu amideswithin the solvent-inaccessible core of the protein are la-beled (Fig. 5C). As shown in Table 1, the molecular dy-namics calculations showed that the values of dNH-O, ∠ NH-

O, and nHOH-O are more widely distributed (larger standarddeviation) for the exposed versus the buried amides. Indeed,for the side-chain–buried 13C-labeled Leu amides, a sharpband is observed at ∼ 1605 cm−1, whose position is essen-tially independent of temperature. Also, the intensity of theband associated with the unlabeled exposed amides near1645 cm−1 is markedly decreased. The position of the 13Cband, together with its temperature independence indicatesthat it is associated with the 13C-labeled amides of the Leuside chains in the solvent-inaccessible interior of the pro-tein.

Analysis of the IR spectra

The amide I� regions of the spectra were fit to a series ofGaussian curves, to allow a more precise determination ofthe positions, intensities, and widths of the various bands.The amide I� region of the spectrum of the unlabeled proteinis dominated by peaks with Gaussian frequencies and

widths as tabulated in Table 1. The protein labeled with13C-Leu shows an additional strong absorption in the 13 Kspectrum with a Gaussian frequency of 1605 cm−1 associ-ated with the 13C-labeled buried amide carbonyl groups(Fig. 5C). Spectral deconvolution additionally reveals a lessintense feature at 1588 cm−1, close to the position expectedfor exposed, 13C-labeled amides. This finding is consistentwith the structure, which shows that Leu-21 lies in a sol-vent-accessible loop between helices 1 and 2 (Fig. 2). Con-versely, the sample labeled with 13C-Ala shows a strongabsorbance at 1588, with a secondary absorbance at 1605cm−1, indicating that most of the amides are accessible tosolvent.

The peak widths provide information concerning the ho-mogeneity of the environment experienced by the amidegroups. At all temperatures, the Gaussian widths of the am-ide I� group of exposed amides are approximately twofoldbroader than for the buried amides (Table 1), presumablyreflecting the heterogeneity of the interactions with solventwithin the 13C-Ala labeled sample. Further, as the tempera-ture is decreased from 290 to 13 K, the width of both bandsdecreases by ∼ 6 cm−1. This behavior is consistent with anoverall decrease in the disorder of the protein and the sur-rounding solvent at low temperatures.

The Gaussian frequencies of the solvent-exposed amidesdepend strongly on temperature, while the frequencies as-sociated with the solvent-inaccessible amides are nearly in-dependent of temperature (Fig. 6A). A plot of the shift inGaussian frequency relative to that observed at 10 K, showsa strong dependence on temperature from 280 K to 160 K;below this temperature a very small change in ��C�O isobserved. Because the carbonyl groups of the Ala residuesare partially exposed to water, one might expect the hydro-gen bonding to the backbone to follow the glass transition ofthe solvent. The absorbance of the OH band, which arisesfrom the HOD in the solvent, is an excellent reporter of thistransition (Fig. 6B, filled diamonds). The glass transitionoccurs at ∼ 160 to 170 K, and coincides with the point at

Table 1. IR and geometric parameters associated with the exposed Ala and buried Leuresidues in �3D�

Sample, temperature�C�O

b

(cm−1) nHOH−O

�C�O width(cm−1)

of dNH−Oc

(Å) of ∠ NH−O

d

(°)

13C-Ala �3D�; r.t.a 1593.8 0.58 29.2 0.33 15.013C-Ala �3D�; 13 K 1587.9 n.a. 23.4 n.a. n.a.13C-Leu �3D�; r.t.a 1606.8 0.12 17.9 0.20 11.813C-Leu �3D�; 13 K 1605.0 n.a. 11.8 n.a. n.a.

aThe IR data are at 290 K, the simulation temperature was 300 K.bThe Gaussian peak position.cThe average standard deviation of dNH−O for the exposed Ala residues (13, 26, 29, 30, 36, 37, 55, 61,62) and the buried Leu residues (11, 28, 42, 56, 67) in the MD simulation. Averages were calculated over2–10 nsec.dThe average standard deviation of ∠ NH−O for the exposed Ala residues (13, 26, 29, 30, 36, 37, 55, 61,62) and the buried Leu residues (11, 28, 42, 56, 67) in the MD simulation.

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which the Ala amide I� band becomes temperature-indepen-dent. This finding provides strong evidence for the interac-tion of water with the solvent-exposed amides.

NMR of �3D

Two parameters were measured by NMR that report ondifferent aspects of the dynamics, hydrogen bonding, andsolvent-accessibility of the amide groups of �3D. The de-viations of the amide proton chemical shifts from the valueexpected for a given amino-acid type in a random coil,��(NH), provide information concerning the strengths ofamide hydrogen bonds, and their interactions with water.Figure 7 illustrates this parameter as a function of positionin sequence for the amides in the three helices of �3D.Within the helical regions, the amide proton chemical shiftsare generally shifted upfield relative to the loops, which isconsistent with the general trend for helices to experience anet negative ��(NH) (Wishart et al. 1991, 1995). Superim-posed on this general trend is a sinusoidal variation in thisparameter, which matches the period of the �-helix (Kuntzet al. 1991; Zhou et al. 1992). However, there is a signifi-cant deviation from this trend in helix 1, at approximatelythe position of the disruption induced by Thr16.

Because a number of different effects can give rise tochanges in chemical shift, we take advantage of the strongsequence and structural similarity between the individualhelices and compute the average value of ��(NH) for allthree of the helices (Fig. 8A). Sites within two residues ofthe ends of the helix were excluded to minimize end effects,and regions that deviated from ideal helical geometries inhelix 1 were also excluded. The resulting curve is welldescribed by a sine wave with a period of 3.61 ± 0.08residues, typical of the �-helical repeat. The mean value of��(NH) for all amides is −0.23 ± 0.04 ppm, which is ingood agreement with the mean upfield shift of −0.2 ppmreported by Wishart et al. (1991). The values of ��(NH)may be converted to a predicted hydrogen bond length(Cierpicki and Otlewski 2001) using either a d−3 (Pardi et al.1983) or a d−1 (Wishart et al. 1991) distance dependenceaccording to the equations:

�� � 19.2d−3 − 2.3 (1)

�� � 19.0d−1 − 9.7 (2)

where d is the hydrogen bond distance in Å from the amideproton donor to the oxygen acceptor, and �� is expressed inparts per minute. Analysis of the data in Figure 8A indicatesthat the most solvent-exposed amide would be expected tohave a hydrogen bond length of ∼ 2.23 Å, whereas the mostsolvent inaccessible amides would have a predicted hydro-gen bond length of 1.95 Å, as determined using equation 2.A similar conclusion was reached by examining the rela-tionship between ��(NH) and dNH-O for helices as de-scribed in Figure 5 of Cierpicki and Otlewski (2001). Thecorresponding values computed using equation 2 are 2.08and 1.91 Å, respectively. Equation 1 agrees with the rangeof distances predicted by the molecular dynamics calcula-tions for helix 1, but it underestimates some of the longerdistances observed for helix 2 (Fig. 2). Equation 2 under-estimates the longest hydrogen bond distances for both he-lices.

Hydrogen-deuterium protection factors (Fig. 7B) providea second parameter defining the environment of the amideprotons. These parameters describe the extent to which hy-drogen-deuterium exchange is slowed relative to the valueexpected for a fully exposed amino acid residue of the sametype. Amides with protection factors matching the overallstability of the protein are able to exchange only when theentire protein unfolds, whereas amides with very low pro-tection factors generally correspond to disordered or mobileloops in the protein. Intermediate behavior corresponds tostructured regions of the protein that are able to locallydistort to allow for exchange with solvent. The amideswithin the helical regions of �3D all showed significantprotection from exchange. Figure 7 compares the protectionfactors for the three helices. It is immediately apparent that

Figure 6. Temperature dependence of the peak position (A) and relativeabsorbance (B) of the peak attributed to the buried 13C-Leu amides (filledsquares) and the exposed 13C-labeled Ala amides (filled circles). The dia-monds in B show the relative absorbance of the OH band at 3500 cm−1.

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the structural distortions observed in helix 1 have signifi-cantly decreased its stability toward hydrogen-deuteriumexchange. The remaining two helices tend to show a sinu-soidal variation in the protection factors, although the pat-tern is less distinct than for the chemical shifts. The devia-tion from sinusoidal behavior is most significant near the Cterminus of helix 3, which we attribute to fraying. The bur-ied amides have protection factors that are well predictedfrom the global stability of the protein (shaded region in Fig.7B), while the more exposed amides exchange more rapidlyby one to two orders of magnitude. The protection factorswere averaged in the same manner as the ��(NH), resultingin a very good correlation with the corresponding plot of theaveraged chemical shift indices (Fig. 8).

Conclusions

The hydration of amide-carbonyl groups on the exposedside of helices has been proposed to play an important role

in defining the stability of helices, and it also exerts a largeeffect on their spectroscopic properties. Here, we use athree-pronged approach to examine the hydration of helicalamides. The dynamics calculations proved to be pivotal toour interpretation of the spectroscopic parameters, and theyhave provided a wealth of molecular detail that should be ofgeneral relevance to understanding the formation and hy-dration of helices. These calculations indicate that watermolecules form hydrogen bonds with the carbonyl groups ofmost exposed amides with fractional occupancies of 0.5 to0.8. The less-than-full occupancy indicates that the free en-ergy of this interaction is relatively weak, on the order ofthermal energy at room temperature. This finding is consis-tent with the relatively small energetic spread in the helicalpropensities, which span ∼ 1 Kcal/mole. Temperature-de-pendent, isotope-edited IR spectroscopy provides a particu-larly convenient method to probe the hydration of amides inhelices. The amide I� band of buried amides is relatively

Figure 7. Amide proton chemical shifts relative to the values expected for a random coil (A) and the protection factors for hydrogendeuterium exchange (B) for helix 1 (top), helix 2 (middle), and helix 3 (bottom) of �3D. The bars are drawn with a 3.6-residue helicalrepeat.

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sharp and its position shows little temperature dependence,indicating that the strength of the hydrogen bond remainsroughly constant from room temperature to 13 K. On theother hand, the hydrogen bonds on the surface of the proteindepend significantly on temperature. Their partial occu-pancy and the heterogeneity of their interactions with waterresult in a broader amide I� vibration at all temperatures. Asthe temperature is decreased, one would expect the occu-pancy and homogeneity of the geometries of the hydrogen-bonded water molecules to increase; this is indeed reflectedin the amide I� band, which sharpens and shifts to lowerenergy, reflecting a strengthening of the hydrogen bonds.

Proton NMR methods provide a complementary methodthat focuses on the amide proton rather than the amide car-bonyl group. Chemical shifts are particularly sensitive tohydrogen bonding and provide a very rapid sequence-spe-cific readout of this interaction. Our molecular dynamicsstudies of hydrogen bonding in �3D complement earlierwork on this subject (Kuntz et al. 1991; Zhou et al. 1992;Cordier and Grzesiek 2002) by explicitly considering therole of water in weakening the hydrogen bonds of amides onthe surface of the protein. Thus, chemical shifts can now be

better interpreted in terms of the occupancy of surface-ac-cessible sites of hydration; as the mean occupancy increasesthe chemical shifts move progressively more upfield. Thesefindings also provide a framework for the understanding ofmore modern methods of detecting hydrogen bonds that relyon the weak J-coupling between the donor amide and theacceptor carbonyl carbon, which provides a direct readoutof particularly strongly hydrogen-bonded interactions(Cordier and Grzesiek 1999, 2002; Cornilescu et al.1999a,b).

An unexpected finding of these studies was the relativelylarge structural and thermodynamic effect of a hydrogenbond between a Thr hydroxyl and a main-chain amide fourresidues prior to it in sequence. This hydrogen bond dis-torted the angle of the N-terminal carbonyl group, which inturn perturbed the helical parameters of the amide precedingit in sequence. Thus, a single Thr hydroxyl had a majorimpact on the structure of 4–7 residues distant in sequence.This disruption was evident in the NMR chemical shifts andmost markedly in the rates of proton exchange. Indeed, theoverall exchange of this helix was significantly more rapidthan the other two helices. This helical disruption was notconsidered in the design of �3D, which suggests that itsstability may be enhanced by either replacing it with anotherpolar residue or by repacking the core to conform to thealtered conformation occasioned by the presence of Thr16.

Materials and methods

Sample preparation

Specifically labeled 13C(1) Leu- and Ala-�3D� samples were pro-duced by growing Escherchia coli strain BL21(DE3) in M9 mini-mal media supplemented with all the amino acids (as the solecarbon source) and 13C(1)-leucine (50 mg/L, Isotec) or 13C(1)-alanine (50 mg/L, Isotec), respectively. Uniformly 15N labeled�3D was produced by growing in M9 minimal media supple-mented with uniformly labeled 15NH4Cl (1 g/L, Isotec) as the solenitrogen source. All �3D variants were purified as described pre-viously (Walsh et al. 1999).

IR spectroscopy

�3D variants for IR studies were resuspended in 5 mM potassiumphosphate buffer in D20 (99.99%, Isotec). The pD was raised to 10with dilute sodium deuteroxide (Isotec) and incubated at 310 K toensure complete exchange of amide protons for deuterons. After 3days at 310 K, the samples’ pD was lowered to 7.0 and lyophilized.IR spectra were acquired with 10 mg/mL of protein in 10 mMpotassium phosphate, D2O-glycerol-d8 (50/50, v/v).

Infrared spectra were obtained with a Bruker IFS 66 Fouriertransform IR instrument. The sample compartment was purgedwith nitrogen to reduce the contribution from water vapor. Thelight levels were monitored using an HgCdTe (MCY) detector.The spectral resolution was 2 cm−1. The spectra were smoothedusing a nine Savitzky-Golay smoothing algorithm. The sampleholder was obtained from Graseby Specac (Smyrna). A 15 mm

Figure 8. Mean chemical shift indices (A) and protection factors (B) as afunction of the position within the regions of helices 1–3. The parametersfrom a total of 14 residues, corresponding to the central 4 turns of thehelices were averaged. The end residues were not included to avoid com-plications associated with end effects. The residues considered span 5 –18(helix 1), 29–42 (helix 2), and 54–67 (helix 3). Additionally, the regionN-terminal to Thr16 (8–11) was also excluded.

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spacer was used between two CaF2 windows in the transmissioncell. The temperature was regulated using an APD closed cycleHelitran cryostat (Advanced Research Systems). The cryostatsample was filled with He gas at atmospheric pressure, which aidsin the transfer of heat from the sample. The outer cryostat windowswere made of CaF2. The inner cryostat windows, which experiencethe temperature gradient, were 2 mm thick and made of ZnSe(Janos Technology). A special holder for these windows was con-structed to minimize strain on the windows from contraction at lowtemperature (Research Instrumentation Shop, University of Penn-sylvania School of Medicine). The temperature was measured witha silicon diode near the sample and the temperature was controlledusing a Model 9650 temperature controller (Scientific Instru-ments). Cryogenic temperature profiles were carried out from highto low. The temperature was measured every 10° and the time forequilibration was 5 to 10 min. The spectra were analyzed for theircomponents using the PeakFit software package (Jandel ScientificSoftware).

Molecular dynamics calculations

All minimization and dynamics calculations were performed usingan in-house version of the program ENCAD (Levitt 1990). Allatoms in both the protein and solvent were explicitly present andthe force-field parameters have been described previously (Levittet al. 1995, 1997). The NMR structure (PDB code 2a3d) (Walsh etal. 1999) was used as the starting structure for the simulations. Thesimulation was performed at neutral pH (Asp, Glu, His, Lys, andArg were all charged). It was initially minimized 500 steps invacuo. Then, it was solvated in a box of water molecules extendingat least 8 Å from any protein atom, resulting in the addition of2893 water molecules and total system size of 9819 total atoms.The box dimensions were then increased uniformly to yield theexperimental density of water at 298 K (0.997 g/mL). Followingthat, the system was equilibrated with 5000 steps of water-onlyminimization, 5000 steps of water-only molecular dynamics at 278K, another 1000 steps of water-only minimization, 500 steps ofprotein-only minimization, and finally 500 steps of conjugate gra-dient minimization of the entire system. Periodic boundary condi-tions were used throughout to minimize edge effects. This was thestarting point for the 10 ns simulation at 298 K using a microca-nonical ensemble (constant NVE). An 8 Å force-shifted non-bonded cutoff was employed, and the nonbonded list was updatedevery five steps. The protocols have been discussed in depth byLevitt and coworkers (Levitt et al. 1995, 1997). Structures weresaved every 0.2 ps for analysis, resulting in 50,000 structures. Theaverages reported in the text were computed from 2–10 ns, or over40,000 structures.

NMR spectroscopy

A 2-mM 15N-�3D sample in 50 mM d3-acetate and 0.05% sodiumazide was used throughout the temperature study. 1H and 15Nchemical shifts (BMRB 4126) of the backbone amide groups weretaken from the 303 K spectrum (Walsh et al. 1999) and extrapo-lated to the other temperatures (278–323 K). Random coil 1Hchemical shifts were taken from (Wishart et al. 1995). Amideproton chemical shift differences were calculated as ��(NH) � �(NH observed) - �(NH random coil).

Experiments were done on a Varian INOVA 600 spectrometerequipped with a triple resonance, triple axis gradient probe. Tentemperature points were collected between 278 K and 323 K. Thetemperature was calibrated using a 100% methanol sample from

278 to 303 K (Raiford et al. 1979). Above 303 K, the temperaturewas calibrated using a 100% ethylene glycol sample (Raiford et al.1979). 1H,15N-HSQC experiments were carried out as described(Zhang et al. 1994). The 1H and 15N carriers were placed at 4.70and 117 ppm, respectively. The spectral widths were 8000 Hz (1H)and 1900 Hz (15N). 1024 and 100 complex points were collectedin the 1H and 15N dimensions, respectively. The data were pro-cessed and analyzed using the program Felix (Biosym Inc.).

Acknowledgments

This work was supported by grants GM48130 (JMV), GM50789(VD), and GM54616 (WFD) from the NIH and the NSF MRSECprogram (WFD).

The publication costs of this article were defrayed in part bypayment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 USC section 1734solely to indicate this fact.

References

Anderson, N.H., Neidigh, J.W., Harris, S.M., Lee, G.M., Liu, Z., and Tong, H.1997. Extracting information from the temperature gradients of polypeptideNH chemical shifts. 1. The importance of conformational averaging. J. Am.Chem. Soc. 119: 8547–8561.

Avbelj, F., Luo, P., and Baldwin, R.L. 2000. Energetics of the interactionbetween water and the helical peptide group and its role in determining helixpropensities. Proc. Natl. Acad. Sci. 97: 10786–10791.

Baker, E.N. and Hubbard, R.E. 1984. Hydrogen bonding in globular proteins.Prog. Biophys. Mol. Biol. 44: 97–179.

Barlow, D.J. and Thornton, J.M. 1988. Helix geometry in proteins. J. Mol. Biol.201: 601–619.

Baxter, N.J. and Williamson, M.P. 1997. Temperature dependence of 1H chemi-cal shifts in proteins. J. Biomol. NMR 9: 359–369.

Belton, P.S. 1994. NMR studies of protein hydration. Prog. Biophys. Mol. Biol.61: 61–79.

Blaber, M., Zhang, X.-J., Lindstrom, J.D., Pepiot, S.D., Baase, W.A., and Mat-thews, B.W. 1994. Determination of �-helix propensity within the contextof a folded protein. J. Mol. Biol. 235: 600–624.

Blundell, T., Barlow, D., Borkakoti, N., and Thornton, J. 1983. Solvent-induceddistortions and the curvature of a-helices. Nature 306: 281–283.

Chakrabarti, P., Bernard, M., and Rees, D.C. 1986. Peptide-bond distortions andthe curvature of �-helices. Biopolymers 25: 1087–1093.

Cierpicki, T. and Otlewski, J. 2001. Amide proton temperature coefficients ashydrogen bond indicators in proteins. J. Biomol. NMR 21: 249–261.

Cordier, F. and Grzesiek, S. 1999. Direct observation of hydrogen bonds inproteins by interresidue 3hJNC� scalar couplings. J. Am. Chem. Soc. 121:1601–1602.

———. 2002. Temperature-dependence of protein hydrogen bond properties asstudied by high-resolution NMR. J. Mol. Biol. 715: 739–752.

Cornilescu, G., Hu, J.-S., and Bax, A. 1999a. Identification of the hydrogenbonding network in a protein by scalar couplings. J. Am. Chem. Soc. 121:2949–2950.

Cornilescu, G., Ramirez, B.E., Frank, M.K., Clore, G.M., Gronenborn, A.M.and Bax, A. 1999b. Correlation between 3hJNC� and hydrogen bond length inproteins. J. Am. Chem. Soc. 121: 6275–6279.

Daggett, V. and Levitt, M. 1992. Molecular dynamics simulations of helixdenaturation. J. Mol. Biol. 223: 1121–1138.

Decatur, S.M. and Antonic, J. 1999. Isotope-edited infrared spectroscopy ofhelical peptides. J. Am. Chem. Soc. 121: 11914–11915.

Garcia, A.E. and Sanbonmatsu, K.Y. 2002. �-Helical stabilization by side chainshielding of backbone hydrogen bonds. Proc. Natl. Acad. Sci. 99: 2782–2787.

Goodman, E.M. and Kim, P.S. 1991. Periodicity of amide proton exchange ratesin a coiled-coil leucine zipper peptide. Biochemistry 30: 11615–11620.

Halle, B. and Denisov, V.P. 2001. Magnetic relaxation dispersion studies ofbiomolecular solutions. Methods Enzymol. 338: 178–201.

Kuntz, I.D., Kosen, P.A., and Craig, E.C. 1991. Amide chemical shifts in manyhelices in peptides and proteins are periodic. J. Am. Chem. Soc. 113: 1406–1408.

Walsh et al.

530 Protein Science, vol. 12

Levitt, M. 1990. ENCAD, Energy Calculations and Dynamics (program). Yeda.Rehovat, Israel.

Levitt, M., Hirshberg, M., Sharon, R., and Daggett, V. 1995. Potential-energyfunction and parameters for simulations of the molecular-dynamics of pro-teins and nucleic acids in solution. Comput. Phys. Commun. 91: 215–231.

Levitt, M., Hirshberg, M., Sharon, R., Laidig, K.E., and Daggett, V. 1997.Calibration and testing of a water model for simulation of the moleculardynamics of proteins and nucleic acids in solution. J. Phys. Chem. B 101:5051–5061.

Luo, P. and Baldwin, R.L. 1999. Interaction between water and polar groups ofthe helix backbone: An important determinant of helix propensities. Proc.Natl. Acad. Sci. 96: 4930–4935.

Manas, E.S., Getahun, Z., Wright, W.W., DeGrado, W.F., and Vanderkooi, J.M.2000. Infrared spectra of amide groups in �-helical proteins: Evidence forhydrogen bonding between helices and water. J. Am. Chem. Soc. 122:9883–9890.

Mattos, C. 2002. Protein-water interactions in a dynamic world. Trends Bio-chem. Sci. 27: 203–208.

Milne, J.S., Mayne, L., Roder, H., Wand, A.J., and Englander, S.W. 1998.Determinants of protien hydrogen exchange studied in equine cytochrome c.Protein Sci. 7: 739–745.

Olivella, M., Deupi, X., Govaerts, C., and Pardo, L. 2002. Influence of theenvironment in the conformation of �-helices studied by protein databasesearch and molecular dynamics simulations. Biophys. J. 82: 3207–3213.

Otting, G. 1997. NMR studies of water bound to biological molecules. J. Prog.Nuc. Magn. Res. Spec. 31: 259–285.

Otting, G., Liepinsh, E., and Wüthrich, K. 1991. Protein hydration in aqueoussolution. Science 254: 974–980.

Pardi, A., Wagner, G., and Wuthrich, K. 1983. Protein conformation and protonnuclear-magnetic-resonance chemical shifts. Eur. J. Biochem. 137: 445–454.

Raiford, D.S., Fisk, C.L., and Becker, E.D. 1979. Calibration of methanol andethylene glycol nuclear magnetic resonance thermometers. Anal. Chem. 51:2050–2051.

Savage, H. and Wlodawer, A. 1986. Determination of water structure aroundbiomolecules using x-ray and neutron diffraction methods. Methods Enzy-mol. 127: 162–183.

Schoenborn, B.P., Garcia, A., and Knott, R. 1995. Hydration in protein crys-tallography. Prog. Biophys. Mol. Biol. 64: 105–119.

Thanki, N., Thornton, J.M., and Goodfellow, J.M. 1988. Distribution of wateraround amino acid residues in proteins. J. Mol. Biol. 202: 637–657.

Vila, J.A., Ripoll, D.R., and Scheraga, H.A. 2000. Physical reasons for theunusual �-helix stabilization afforded by charged or neutral polar residuesin alanine-rich peptides. Proc. Natl. Acad. Sci. 97: 13075–13079.

———. 2001. Influence of lysine content and ph on the stability of alanine-based copolypeptides. Biopolymers 58: 235–246.

Walsh, S.T.R., Cheng, H., Bryson, J.W., Roder, H., and DeGrado, W.F. 1999.The solution structure and dynamics of a de novo designed three-helixbundle protein. Proc. Natl. Acad. Sci. 96: 5486–5491.

Walsh, S.T.R., Lee, A.L., DeGrado, W.F., and Wand, A.J. 2001a. Dynamics ofa de novo designed three-helix bundle protein studied by 15N, 13C, and 2HNMR relaxation methods. Biochemistry 40: 9560–9569.

Walsh, S.T.R., Sukharev, V.I., Betz, S.F., Vekshin, N.L., and DeGrado, W.F.2001b. Hydrophobic core malleability of a de novo designed three-helixbundle protein. J. Mol. Biol. 305: 361–373.

Wishart, D.S., Sykes, B.D., and Richards, F.M. 1991. Relationship betweennuclear magnetic resonance chemical shift and protein secondary structure.J. Mol. Biol. 222: 311–333.

Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S., and Sykes, B.D. 1995. 1H,13C, and 15N random coil NMR chemical shifts of the common amino acids.I. Investigations of nearest-neighbor effects. J. Biomol. NMR 6: 67–81.

Zhang, O., Kay, L.E., Olivier, J.P., and Forman-Kay, J.D. 1994. Backbone 1Hand 15N resonance assignments of the N-terminal SH3 domain of drk infolded and unfolded states using enhanced-sensitivity pulsed field gradientNMR techniques. J. Biomol. NMR 4: 845–858.

Zhou, N.E., Zhu, B.-Y., Sykes, B.D., and Hodges, R.S. 1992. Relationshipbetween amide proton chemical shifts and hydrogen bonding in amphipathic�-helical peptides. J. Am. Chem. Soc. 114: 4320–4326.

Hydration of amides in helices

www.proteinscience.org 531