Environment-dependent long-rangestructural distortion in a temperature-sensitive point mutant

Jannette Carey,1* Brian Benoff,2 Balasubramanian Harish,1

Lara Yuan,1 and Catherine L. Lawson2*

1Chemistry Department, Princeton University, Princeton, New Jersey 085442Department of Chemistry & Chemical Biology, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854

Received 19 September 2011; Revised 25 October 2011; Accepted 27 October 2011DOI: 10.1002/pro.759Published online 4 November 2011 proteinscience.org

Abstract: Extensive environment-dependent rearrangement of the helix-turn-helix DNA recognition

region and adjacent L-tryptophan binding pocket is reported in the crystal structure of dimeric

E. coli trp aporepressor with point mutation Leu75Phe. In one of two subunits, the eight residuesimmediately C-terminal to the mutation are shifted forward in helical register by three positions,

and the five following residues form an extrahelical loop accommodating the register shift.

In contrast, the second subunit has wildtype-like conformation, as do both subunits in anisomorphous wildtype control structure. Treated together as an ensemble pair, the distorted and

wildtype-like conformations of the mutant apoprotein agree more fully than either conformation

alone with previously reported NOE measurements, and account more completely for its diversebiochemical and biophysical properties. The register-shifted segment Ile79-Ala80-Thr81-Ile82-Thr83

is helical in both conformations despite low helical propensity, suggesting an important structural

role for the steric constraints imposed by b-branched residues in helical conformation.

Keywords: hydrophobic brace; rotamer; cooperativity; crystal packing; ILV cluster

IntroductionThe tryptophan repressor of Escherichia coli (TrpR)

has served as an important early example of the

complexity of relationships among protein flexibility,

stability, and function. The TrpR dimer has extraor-

dinary thermal stability, unfolding cooperatively to

monomers in vitro with a midpoint temperature of

�92�C.1 Yet many complementary lines of experi-

mental evidence indicate that the helix-turn-helix

region (HtH) that contacts DNA is conformationally

mobile even in the presence of its ligands L-trypto-

phan and DNA. The earliest evidence came from

crystallographic studies showing that HtH helices D

and E (Fig. 1) can adopt different positions relative

to the dimeric protein core (helices A, B, C, and F of

both subunits), independent of L-tryptophan occu-

pancy.2 NMR studies by Jardetzky and coworkers3–7

demonstrated that segments D and E are helical in

solution on a nanosecond, but not a millisecond, time-

scale even in the presence of L-tryptophan. Thermo-

dynamic analysis of TrpR binding to L-tryptophan

and to DNA8 revealed very large heat capacity

changes, the signature of protein surface burial.9

Both crystallography and NMR showed that DNA

binding is accompanied by conformational adjustments

Additional Supporting Information may be found in the onlineversion of this article.

Brian Benoff current address is Middletown Board of Education,900 Nutswamp Road, Middletown, NJ 07748.Lara Yuan current address is Boston Biomedical Consultants,1000 Winter Street, Waltham, MA 02451.

Grant sponsor: NIH; Grant number: GM21589; Grant sponsor:National Science Foundation; Grant number: OISE0853423

*Correspondence to: Jannette Carey, Chemistry Department,Princeton University, Princeton, NJ 08544. E-mail: jcarey@princeton.edu or Catherine L. Lawson, Department ofChemistry & Chemical Biology, Rutgers, The State University ofNew Jersey, 610 Taylor Road, Piscataway, NJ 08854. E-mail:cathy.lawson@rutgers.edu

Published by Wiley-Blackwell. VC 2011 The Protein Society PROTEIN SCIENCE 2012 VOL 21:63—74 63

in HtH helices D and E.10,11 Further indications of

HtH mobility derive from proteolytic sensitivity in

helix D,12–14 molecular dynamics simulations,15–18 and

dramatic conformational rearrangement of the HtH

region in a domain-swapped crystalline TrpR gel.19

A genetic screen designed to identify mutations

conferring temperature-sensitivity in TrpR function

led to isolation of the point mutant L75F.20 In wild-

type (wt) TrpR, the partially solvent-exposed side-

chain of Leu75 at the C-terminus of helix D contacts

residues of the HtH and the dimeric core. Compared

to wt, the L75F mutant protein with Phe75 has

�10-fold weaker affinity for L-tryptophan, 2- to 5-

fold weaker affinity for operator DNA, and slight

increases in helix content and stability toward urea

denaturation.20 Although NMR spectra indicate that

the average solution structure of L75F apoTrpR

resembles wt apoTrpR, long-range effects on dynam-

ics are evident from significant changes in NOE pat-

terns and amide-proton exchange rates.20 Very large

NMR chemical-shift differences are detected for resi-

dues �25 A distant from Phe75, much too far away

to be ascribed to ring-current effects, and seemingly

inconsistent with the wt-like average solution struc-

ture.21 Measurements of apoTrpR backbone amide-

bond dynamics by 15N relaxation indicate that helix

D is more ordered in L75F than in wt, and that he-

lix E is less ordered.22

We initiated crystallographic study of the L75F

apoTrpR mutant to investigate the unusual biophys-

ical properties conferred by the L75F point muta-

tion. We report here the structure elucidation and

analysis of isomorphous crystals of L75F and wt

apoTrpR, which enable resolution of effects of the

L75F mutation from effects of crystal packing and

permit comparison with the NMR data.

Results and Discussion

Isomorphous wt and L75F apoTrpR

crystal structures

Structures of wt and L75F apoTrpR were obtained

from monoclinic crystals grown under identical con-

ditions and analyzed in parallel (see Methods). Each

structure consists of one complete homodimer with

two crystallographically independent subunits. Sum-

mary crystal, data collection, and refinement statis-

tics are provided in Table I.

In each structure the two subunit chains differ

strongly in relative order as judged by main-chain

atomic displacement parameters, particularly in

N-terminal arm and HtH regions [Fig. 2(A,B)]. The

nonequivalent HtH regions are designated here as

HtH1 and HtH2. The asymmetry is a consequence of

differences in local crystal packing environments

(Supporting Information Fig. S1). HtH1 has exten-

sive contacts with the N-terminal arm of a neighbor-

ing dimer, whereas HtH2 is largely free of crystal

contacts. The N-terminal arm-HtH1 contact buries

�900 A2 of total accessible surface area and resem-

bles the arm-HtH interaction between TrpR dimers

bound to DNA as well as interdimer contacts seen in

other TrpR crystal structures (Fig. 3). The relative

orientations of the interacting dimers and the extent

of arm-to-HtH contact differ considerably among

these structures, but in each case the N-terminal

arm Tyr7 phenol ring fills a shallow, largely hydro-

phobic depression lined by residues within or near

the HtH (Leu61, Leu62, Ser86, Leu89, Lys90, and

Arg97). The presence of arm-HtH interactions in mul-

tiple TrpR crystal structures lends strong support to

the suggestion that TrpR N-terminal arms mediate

higher-order association of dimers in solution.25–27

The two subunits of the wt dimer have nearly

identical conformations [Fig. 2(A)], but the two L75F

subunits differ substantially in the conformations of

their HtH regions [Fig. 2(B)]. The better-ordered

HtH1 of L75F [Fig. 2(B)] participates in the arm-

HtH tandem-like contact [Fig. 3(C)], (Supporting

Information Fig. S1) and has a wt-like fold. The more

poorly ordered HtH2 of L75F has a substantially

deformed helix E and D-E turn. Because the HtH2

region is relatively free of crystal contacts in both

Figure 1. wt holoTrpR,2 showing the arrangement of

helices (white subunit: A–F; dark subunit: a–f) and

L-tryptophan ligands (black sticks, dotted surface). Leu 75

lies within the DNA-binding helix-turn-helix region (white

subunit: D,E; dark subunit: d,e) and is shown as a skeletal

model indicated with black arrows. An interactive view is

available in the electronic version of the article.

64 PROTEINSCIENCE.ORG Long-Range Distortion Permitted by Point Mutation

structures, the altered conformation in the L75F

structure is likely a consequence of the mutation.

Comparison of wt and L75F HtH1 regions

Within the better-ordered HtH1 region the wt and

mutant structures are only subtly different from each

other. The Phe75 phenyl ring occupies the same posi-

tion as the Leu75 isobutyl group in the wt structure

[Fig. 2(C)]. Accommodation of the slightly longer,

bulkier Phe75 is achieved not locally but through a

�1 A rigid-body shift of the entire HtH1 [Fig. 2(C)].

The numerous contacts made by the side-chain of res-

idue 75 with residues of helix C are conserved.

The L-tryptophan binding site adjacent to HtH1

has a slightly larger pocket volume in L75F (316 A3;

see Methods) when compared with wt (302 A3), but

these are both much larger than in previously deter-

mined crystal structures for apoTrpR (178 A3) and L-

tryptophan-bound TrpR (246 A3).2,28 The variation in

L-tryptophan pocket volume results directly from flexi-

bility of the HtH helices, which adopt different posi-

tions in different crystal structures.2 Both wt and

L75F pockets contain partially ordered solvent. The

L75F binding site holds in addition a tris cation

((HOCH2)3CNH3þ) bound with partial occupancy; the

tris amino nitrogen and bulky central carbon sit

roughly at L-tryptophan a-amino nitrogen and a-car-

bon positions, respectively [compare Fig. 1 and Fig.

2(B)]. The presence of tris in only the L75F structure

suggests that the mutant may be more permissive

than wt to entry of molecular entities bulkier than the

native ligand. Although the binding of tris adjacent to

HtH1 is unique to the L75F structure, it is unlikely

that its presence influences HtH2 secondary structure,

since there is no cooperativity between the two L-tryp-

tophan binding sites of the TrpR dimer.8,20,29

Comparison of wt and L75F HtH2 regionsWithin the HtH2 region the two structures differ

more substantially. Helix D of the mutant is shifted

essentially as a rigid body by �3 A relative to wt

[Fig. 2(D)]. In addition, the residues that belong to

the interhelical turn and helix E in the wt structure

undergo a remarkable conformational change in the

mutant (Fig. 4). Residues 76–83 form an alternative

helix (E0) with the first turn in 310 conformation.

Mutant helix E0 occupies approximately the same

position as wt helix E residues 79–86; in other words,

the mutant helix is shifted out-of-register by three

residues relative to wt (Fig. 4). In the register-shifted

conformer, Ile82 takes the position occupied in wt by

Gly85. In the wt conformer the absence of a side-

chain at Gly85 accommodates the L-tryptophan

indole18,30; therefore, L-tryptophan binding would be

incompatible with the distorted mutant conformation.

In the mutant, the five residues following helix

E0 form a large loop, whereas in wt the same resi-

dues form the central part of helix E [Fig. 2(D)]. The

entire loop is heavily solvated and makes no crystal

contacts. The largest Ca-atom position shift between

wt and L75F structures is �10 A and is found in

this segment at Ser86, 11 residues distant in pri-

mary structure from the mutation site. The sequence

of loop residues 85–88, GlySerAsnSer, is turn-like

Table I. wt and L75F apoTrpR Crystal Structure Statistics

Space groupwt apoTrpR L75F apoTrpR

P21 P21

Unit cell lengths (A) a ¼ 35.7, b ¼ 54.3, c ¼ 56.9 a ¼ 35.7, b ¼ 54.1, c ¼ 55.6Unit cell angles (�) a ¼ 90, b ¼ 103.2, c ¼ 90 a ¼ 90, b ¼ 100.1, c ¼ 90Asymmetric unit content 2 subunits (1 dimer) 2 subunits (1 dimer)Solvent fraction 0.48 0.48Radiation source NSLS X26C NSLS X26CWavelength (A) 1.1 1.1Measured Reflections 176,611 103,879Unique reflections 24,536 28,206Resolution range (A) 50–1.67 (1.73–1.67) 50–1.58 (1.64–1.58)hI/ri 13.2 (4.5) 12.1 (5.5)Completeness 0.989 (0.948) 0.979 (0.877)Rmerge 0.066 (0.597) 0.073 (0.496)PDB id 3SSW 3SSXResolution range (highest resolution shell) (A) 24–1.67 (1.71–1.67) 27–1.58 (1.62–1.58)Reflections used in refinement 23,235 26,533Completeness for range (%) 93.8 92.4Rwork 0.176 (0.237) 0.176 (0.249)Rfree 0.219 (0.279) 0.219 (0.290)Protein atoms 1626 1684Solvent atoms 201 238RMS deviation, bonds (A) 0.02 0.02RMS deviation, angles (�) 1.49 1.79Residues with allowed f,w (%)a 100 100Residues with favored f,w (%)a 100 99.5

Carey et al. PROTEIN SCIENCE VOL 21:63—74 65

with low propensity for both helix and strand (Sup-

porting Information Fig. S3).

Residues 89–92 are in roughly the same position

and orientation in both mutant and wt structures,

forming the last turn of helix E in wt and a single-

turn helix (E00) in L75F. Mutant helices E0 and E00

are tip-to-tip and nearly coaxial (offset �30�). Atom

geometries and distances imply that the two seg-

ments are linked by a single helical-like backbone

hydrogen bond between Ile82 and Leu89, and by a

backbone hydrogen bond between Thr83 and Lys90

bridged by a water oxygen. Although the main-chain

conformation of Ser88 is extended beta rather than

helical, its carbonyl group makes the only i, i þ 4

backbone hydrogen bond within helix E00, to the

Ala92 amide. The Ser88 side-chain hydroxyl oxygen

can also form a hydrogen bond with the Ala91 am-

ide, providing an N-terminal cap31 for the short E00

helix. There are no side-chain interactions between

helices E0 and E00 instead, residues of both helical

segments participate in hydrophobic contacts with

residues of surrounding helices as discussed further

below.

Resolving effects of crystal contacts

and mutationThe dramatic difference in conformations of the two

chains of the L75F mutant apoTrpR dimer raises the

question of whether the distortions in HtH2 arise

from mutation or from crystal packing. When the

two HtH regions are superimposed on each other

within the context of the crystal lattice, the rela-

tively open environment of the deformed HtH2

appears to offer no steric hindrance to adoption of

the wt-like conformation. A refinement test designed

to investigate the possibility that wt-like conforma-

tion might be adopted by at least a small percentage

of L75F apoTrpR dimers at the HtH2 position within

the crystal gave a clear negative result (see Meth-

ods). In contrast, the relatively restrictive HtH1

Figure 2. Comparison of isomorphous apoTrpR crystal structures. (A) wt apoTrpR and (B) L75F apoTrpR are displayed in

ribbon representation with colors reflecting main-chain atomic displacements (<25 A2: dark blue; >55 A2: red). The two

independent helix-turn-helix regions are indicated as HtH1 and HtH2. Black arrows and skeletal side-chains indicate L75F

point mutation positions. A tris cation occupying one L-tryptophan binding pocket in the L75F apoTrpR structure is also

shown (compare dotted surface for tris in (B) and for L-tryptophan in Fig. 1). In (C) and (D), HtH regions of the two structures

are shown overlaid after overall least-squares fit of all dimer Ca atoms. (C) HtH1 (wt: gray; L75F: cyan with selected residue

labels and Phe 75 in magenta). (D) HtH2 (as in panel C except L75F is colored orange). An interactive view is available in the

electronic version of the article.

66 PROTEINSCIENCE.ORG Long-Range Distortion Permitted by Point Mutation

lattice environment, which includes the tandem-like

arm-HtH contact, cannot accommodate the deformed

conformation owing to severe clashes. These obser-

vations indicate that the HtH conformation of the

L75F mutant depends on the environmental context,

i.e., whether or not there is contact with a neighbor-

ing dimer in the crystal lattice. Analogously, the

HtH2 lattice environment of wt apoTrpR offers no

steric hindrance to adoption of a distorted conforma-

tion. Thus, the conformational difference between wt

and mutant HtH2 conformations can be attributed

directly to the L75F mutation.

To better understand how the L75F mutation

enables the conformational change, side-chain pack-

ing interactions in the apoTrpR structures were ana-

lyzed. The two alternative packing arrangements

can be described as differing in the register of the

helical Ile79-Ile82 segment relative to invariantly

Figure 3. TrpR N-terminal arm-HtH interactions in (A) tandem trpR-DNA complex crystal structure (PDBid 1trr),23 (B) TrpR

crystal structure (PDBid 1wrp),28 (C) apoTrpR crystal structure (this work; PDBid 3ssw). Each panel shows a pair of trpR

dimers involved in an arm-HtH interaction (HtH: green subunit; arm: orange subunit; in the tandem complex only (panel A), a

second symmetry-related arm-HtH interaction is also shown). In the top half of each panel, the first (blue/green) dimer view

orientation is held constant to highlight the range of positions occupied by the second (orange/pink) dimer; Pro6 and Tyr7

residues of the N-terminal arm are displayed in red on each subunit (L-trp: black; DNA: thin wire). In the bottom half of each

panel, details are shown for each interaction, with arm residues Pro6 and Tyr7 (carbon: orange; nitrogen: blue; oxygen: red)

and interacting HtH residues (carbon: green; nitrogen: blue; oxygen: red). In apoTrpR (panel C), an arm-HtH interaction is

made only to HtH1; although a second N-terminal arm (green subunit) is proximal to HtH2 (orange subunit), it is not visible in

electron density at Pro6, Tyr7 positions. The local environment difference between HtH1 and HtH2 provides an explanation for

the selective permissiveness to conformational change observed in the isomorphous L75F mutant structure. Arm-HtH

interactions closely similar to those seen in the tandem trpR-DNA complex (A) are also observed in a tetragonal crystal form

of holoTrpR24 (structure not shown; PDBid 1ZT9).

Figure 4. Structure-based sequence alignment of distorted L75F and wt subunits. Residues 57–93 of each 107-residue

subunit are shown with helix assignments. Boxes enclose residues in helical conformation as judged by main-chain f,wangles. Gray shading indicates 310-helix conformation. Lower-case denotes residues in turns. Dots indicate positions usually

occupied by hydrophobic residues in helix-turn-helix DNA binding proteins28 (‘‘hydrophobic brace’’).

Carey et al. PROTEIN SCIENCE VOL 21:63—74 67

positioned residues of the L-tryptophan binding

pocket: Ile57 and Val58, which are located in the

C-terminal half of helix C, and Leu89, located near

the C-terminus of helix E or within helix E00 (Fig. 5).

In wt and wt-like mutant conformations, Leu/Phe75

and helical residues Thr81 and Ile82 pack against

Ile57 [Fig. 5(A,B)]. In the distorted mutant conforma-

tion, the helix-register shift and 310 conformation

result in Ile79 occupying the wt position of Ile82, and

Ile82 occupying the wt position of Gly85 [Fig. 5(C)].

In the distorted conformation only, Ile79 joins the

cluster of packed residues. Thus, the alternative pack-

ing of two segments with relatively invariant local

structures, 79–82 and 57–58, links the burial of Ile79

to the looping out of residues 84–88 from helix E.

As expected, the solvent-accessible surface areas

of the two packing arrangements differ significantly.

When the whole chain or only the HtH regions are

considered, the distorted conformer of apo L75F

TrpR buries slightly less hydrophobic as well as total

surface area than the wt-like conformer, and both

conformers bury slightly more surface area than wt

apoTrpR (Supporting Information Table S1). How-

ever, when only the alternatively packed b-branched

segments are considered, the distorted L75F con-

former has hydrophobic and total surface area burial

that is approximately 2-fold more efficient than wt

or wt-like L75F. Although the crystal structures

indicate that both conformations are accessible to

L75F apoTrpR, the enhanced local surface burial of

L75F may explain why the distorted conformation is

favored for the mutant in the absence of crystal con-

tacts. Differences in solvent-accessible surface areas

cannot be used directly to assess the relative stabil-

ities of the two conformers because crystallization is

an inherently hysteretic process and thus crystal

structures need not reflect equilibrium distributions

of conformers. On the other hand, because the differ-

ences in structure between the two conformers of

L75F are strictly local, and appear to be controlled

by the alternative packing of these b-branched seg-

ments, the relative stabilities of the two conformers

may also reflect the local packing efficiency of these

segments rather than the global packing efficiency.

Comparison with NMR dataNMR data for L75F TrpR clearly indicate the long-

range nature of the effects of the L75F mutation.

Although early NMR spectra were fully consistent

with a wt-like average structure, the number of

NOESY cross-peaks was much larger than in wt

spectra acquired under the same conditions.20

ROESY spectra ruled out spin diffusion as a trivial

cause of the NOESY results.20 The rate of deuterium

exchange of unresolved amide protons was �50%

slower than in wt, as were exchange rates for the

resolved indole protons of both Trp19 and Trp99.20

All of the proton exchange data were adequately fit

by single-exponential decay functions, indicating

generally similar rate-limiting steps for exchange in

the wt and mutant proteins. Two well-resolved,

slowly-exchanging amide protons were detected in

1D spectra of the mutant only, as well as a promi-

nent NOESY crosspeak between them20; these were

later assigned to Met42 and Leu43 in the B-C

turn.21 In addition, a prominent HSQC crosspeak

unique to the mutant was detected20 that was later

assigned to the Thr44 amide.21

Full assignment of L75F apoTrpR21 revealed

extremely large chemical shift changes for residues

up to �26 A from Phe75, far too distant to be

explained by effects of the ring current of the new

aromatic residue. Furthermore, many distant resi-

dues were very strongly shifted while others closer

to the mutation site were not. Among amide nitro-

gens assigned in both mutant and wt, the largest15N chemical shift differences were found for Thr81,

Ile82, and Thr83, which shift upfield by 2.03, 3.90,

Figure 5. Alternative side-chain packing in the L75F apoTrpR mutant. (A) wt apoTrpR (B) L75F apoTrpR, wt-like subunit (C)

L75F apoTrpR, distorted subunit. In all three panels, side-chains of selected residues are represented as space-filling models;

the backbone is represented as a white tube. The indole ring position of the L-trp binding site is indicated in (A) and (B) as a

semitransparent gray surface (in front of G85, V58, and L89 in this view). The side-chain for Thr81, discussed in the main text,

was omitted from the images for clarity. In panel A and B views, Thr81 sits directly in front of Ile82.

68 PROTEINSCIENCE.ORG Long-Range Distortion Permitted by Point Mutation

and 4.42 ppm, respectively; the largest shifts for

assigned amide nitrogens closer to the mutation site

are 1.32 ppm upfield for Asn73 and 1.75 ppm down-

field for Gly76. For comparison, the distances from

Phe75 to each of these residues on the wt-like side

of the L75F crystal structure reported here are

Thr81, 10.4 A; Ile82, 10.2 A; Thr83, 14.0 A; Asn73,

5.9 A; and Gly76, 3.8 A. Examples of residues that

are similarly distant from Phe75 as residues 81–83

but experience little or no change in chemical shift

include Gly78, 8.7 A, �0.17 ppm; and Glu70, 8.3 A,

�0.08 ppm. The chemical-shift results in the L75F

HtH region were previously rationalized as reflecting a

global response to the mutation with effects at nearby

residues complicated by ring-current shifts.21 The ring

current was assumed to extend as much as 10 A from

Phe75 to include potential anisotropic effects. Never-

theless, very significant 15N chemical-shift changes

(�1–2 ppm upfield for backbone amides) as distant as

�28 A away in the same subunit and �26 A away in

the other subunit could not be explained.

The average NMR structure of L75F apoTrpR

derived from NOESY restraints is very similar to

that of wt TrpR, as are all the individual members

of the family of accepted structures.21 Like all NMR

structures of TrpR solved to date, the dimeric core of

L75F apoTrpR (helices A, B, C, and F) is well-

constrained by the data. In contrast, considerable

variation in the HtH regions of L75F was attributed

to instability of these domains, following the inter-

pretation of more extensive data available for wt

apoTrpR.3,5 When compared with the dimeric core,

the L75F and wt HtH regions share a general pau-

city of NOEs, limited protection of amides from

exchange, and smaller chemical-shift deviations

from random coil, indicating that they are much less

well-defined. However, other NMR measures indi-

cate apparently more helical structure for the HtH

of L75F than for wt. L75F residues 67–83 have

several i, i þ 3 NOEs and two i, i þ 4 NOEs between

a-carbon and backbone amide protons that are

absent in wt, as well as larger helical chemical shift

deviations for a-carbons and a-protons.21 These data

provide the restraints that result in models for L75F

apoTrpR that are more helical in the HtH region

than corresponding models for wt.

Although the environment-dependent distortion

in the L75F crystal structure suggests that both con-

formations might also occur in solution, none of the

HtH region conformations in the family of accepted

NMR solution structures for L75F apoTrpR (PDB id

2xdi)21 closely resembles either conformation in the

crystal structure. This finding prompted evaluation

of both wt-like and distorted conformations against

NOE-based distance restraints. Of 728 measured

NOEs, only ten conflict with either one or both crys-

tal structure conformations, with inferred interpro-

ton distances that are too long to generate an NOE

of the measured strength (Table II). Significantly,

the discrepancies are not found throughout the mol-

ecule but involve exclusively residues involved in

the alternative packing depicted in Figure 5. Many

of these discrepancies do involve residues relatively

close to the Phe75 aromatic ring, but ring-currents

that can affect chemical shift positions do not affect

relaxation processes such as NOEs.

Of the 10 identified NOEs, five are consistent

with the distorted conformation but inconsistent

with wt-like conformation. In particular, two strong

NOEs with upper limit distances of 4.5 A from Ile79

to Gly76 and to Ala77 are much better accounted for

by the distorted structure. Conversely, 2 of the 10

NOEs are fully consistent with wt-like conformation

but inconsistent with distorted conformation. Thus,

neither structure alone can account for all the data.

The final 3 of the 10 NOEs involve residue pairs

Phe75-Ala80, Gly76-Ile79, and Asn87-Leu89; neither

wt-like nor distorted conformations have interproton

distances sufficiently short to satisfy the correspond-

ing restraints. However, an intermediate conforma-

tion representing a transition between the two crys-

tallographic conformations might reasonably be

expected to bring each of these residue pairs into

sufficiently close proximity to yield an NOE.

NOE strength, though proportional to distance,

is not necessarily proportional to population or dura-

tion; thus, NOEs can over-represent transitory close

encounters. Indeed, van Gunsteren32 has developed

an approach that uses NOE-derived restraints as

time-averaged bounds. In this method the ensemble as

a whole satisfies all the observed NOEs, rather than

the conventional treatment that requires every struc-

ture in the ensemble to satisfy all restraints. If the

HtH region of L75F apoTrpR alternates between two

conformations in solution, each conformation would

account for only some of the NOEs, and the structure

calculated by the conventional approach would not

reflect these dynamics, but present only an apparently

disordered region. This situation appears to best

account for the NMR results on L75F apoTrpR.

Reanalysis of NMR evidence for L75F apoTrpR

thus suggests that more than one conformation

must be populated in solution to account for all the

data. The highly nonuniform patterns of NMR

changes include those that are predicted if the dis-

torted crystal conformer of L75F were present in

solution. A recent analysis of 15N backbone relaxa-

tion data for L75F apoTrpR22 indicates that the

spin–spin relaxation of Ile82 is strongly affected by

an exchange process, supporting the existence of a

conformational transition involving the HtH region.

The relaxation data also indicate enhanced flexibil-

ity for residues 84–88 in L75F relative to wt. The

picture that emerges is that in solution, L75F

apoTrpR samples conformations that likely include

both conformations observed in the crystal structure.

Carey et al. PROTEIN SCIENCE VOL 21:63—74 69

The transition between the two crystallographic con-

formations could be accomplished by relatively mod-

est chain excursions compared with conversion to

the domain-swapped gel form that both wt and L75F

TrpR undergo.19

In addition to accounting for NOEs in the imme-

diate vicinity of the mutation, the presence of the

distorted structure in solution would provide a

highly satisfactory explanation for the effects

detected by NMR involving the distant amide groups

of Met42, Leu43, and Thr44. In wt apoTrpR and the

wt-like side of L75F apoTrpR, these residues are

closely approached by the guanidinium group of

Arg84, whereas in the distorted L75F conformation

the looping-out of segment 84–88 between helices E0

and E00 moves Arg84 away and brings several

uncharged residues nearby (Thr81, Ile82, Ser86,

Asn87). This change represents a substantial pertur-

bation of the electronic environment that could read-

ily affect nearby amide protons. Furthermore, since

Leu43 methyl protons display NOEs to Trp19 indole

ring protons, the change might be responsible for

the large chemical shift changes experienced by all

the hydrophobic residues on the Trp19 face of helix

A in the L75F mutant, as well as the blue-shifting of

the fluorescence emission spectrum attributed to

Trp19 (see discussion below).20 All of these puzzling

perturbations detected at so many residues far

removed from the site of mutation have not other-

wise been explained by any NMR structures or

relaxation analyses conducted to date.

Comparison with biophysical data

A large body of data that has accumulated on L75F

apoTrpR can be partly rationalized by the structures

and analysis reported here.

The urea denaturation midpoint monitored by

circular dichroism (CD) is slightly higher than for

wt (�7.0 M vs. �6.5 M), but the thermal midpoint

assessed by differential scanning calorimetry (�91�C)

is unaltered.20 The heat capacity change upon dena-

turation is similar to that of wt (�90 kcal/mol), and

the ratio of van’t Hoff to calorimetric enthalpy is near

one for both proteins, indicating a two-state unfolding

process.20 These data are consistent with the general

picture of TrpR as a highly stable but reversibly fold-

ing dimer.33,34 The interdependent tertiary and qua-

ternary organization of the intertwined central core

is expected to be relatively insensitive to structural

changes in the more peripheral HtH DNA-binding

domains, consistent with local control of HtH stability

based on the packing data. The independence of the

HtH domains from the central dimer fold is dramati-

cally illustrated by the finding that chymotrypsin

cleavage of wt apoTrpR dimers within the HtH yields

four fragments that reassociate spontaneously to

regenerate the native NMR spectrum.14

Although apoTrpR stability is largely unaltered

by the L75F mutation, the CD spectrum shows an

increase in helix content compared to wt from �77

to �85% of residues.20 However, the structures show

that WT apoTrpR has two more residues in helical

conformation than the distorted conformer, in appa-

rent opposition to the CD results, although the effects

of helix length and distortion can complicate interpre-

tation of the fraction of helical residues.35 On the

other hand, the increased helical CD intensity and

slightly higher resistance to urea denaturation are

consistent with the modest improvement in relative

order seen in the crystal (Fig. 2). Both proteins reach

�87% helix content upon titration with trifluoroetha-

nol,20 implying that even in the absence of cosolvent

L75F TrpR has nearly the maximal helix content per-

mitted by its primary structure. Only the domain-

swapped crystalline gel formed by wt or L75F TrpR

in isopropanol has higher helix content (�89% as cal-

culated from the structure), due to conversion of the

C-D and D-E turns to helical conformation.19

Table II. Comparison of NOEs and Inferred Interproton Distances for L75F apoTrpR

NOEs Inferred H-H distancesL75F apoTrpR in solutiona L75F apoTrpR crystal structureb

Proton 1 Proton 2NOE distanceupper limit (A)

wt-likeconformation (A)

Distortedconformation (A)

Phe 75 Hb Ile 79 Hc2 6.5 11.8–14.5 4.5–5.9Phe 75 Hd1 Ile 79 Hc2 8.3 11.9–13.5 7.9–8.5Gly 76 H Ile 79 Hb 4.5 12.2 4.4Gly 76 Ha Ile 79 H 5.5 8.7–9.6 4.8–5.1Ala 77 Ha Ile 79 H 4.5 6.5 3.7Ile 57 Hc2 Thr 81 Hc2 5.5 3.6–6.6 6.7–9.9Phe 75 H Ala 77 Hb 6.0 4.6–6.3 8.8–9.6Phe 75 Hb Ala 80 Hb 6.5 11.5–13.5 8.4–9.8Gly 76 H Ile 79 Hc1 5.5 10.4–11.4 6.4–7.2Asn 87 Ha Leu 89 Hd1 6.7 7.1–9.3 8.5–11.5

a NOE restraints for PDB id 2xdi.21

b Interproton distances or distance ranges were measured between predicted hydrogen atom positions in the L75F apoTrpRcrystal structure (PDB id 3SSX). Bold ranges indicate values consistent with the observed NOE.

70 PROTEINSCIENCE.ORG Long-Range Distortion Permitted by Point Mutation

The intensity-weighted fluorescence emission max-

imum is blue-shifted by �2.3 nm in L75F apoTrpR rela-

tive to wt, mainly due to changes at the red edge of the

spectrum, with an overall increase in intensity of

�14%.20 With excitation at 280 nm, emission is con-

fined almost entirely to the two native Trp residues at

positions 19 and 99. Based on analysis of the NMR

results,21 the spectral changes could be assigned to an

altered electronic environment at Trp19 despite its

being �25 A away from Phe 75 in the same subunit and

�15 A away in the other subunit in wt conformation.

However, the distorted conformation of L75F better

explains the fluorescence results as reflecting the prox-

imity of Trp19 to Leu43 in the L-trp binding pocket,

where the electronic environment is strongly affected

by the alternating proximity of Arg84. The altered L-trp

binding affinity of L75F20 is also consistent with occlu-

sion of the binding pocket by the distortion.

Significance of b-branched residuesin helical segments

In the course of comparing the 20 accepted NMR

structures of L75F apoTrpR with the crystal struc-

ture reported here, the isolated HtH regions of the

forty subunits of the NMR models and the two crys-

tal subunits were superimposed on each other inde-

pendently of their positions with respect to the dimer

core. Residues 80–82 were found to adopt a highly sim-

ilar conformation in all structures. This result was

unexpected considering the limited set of NMR con-

straints available in the HtH region, and prompted a

more detailed evaluation based on the variation in

main-chain phi, psi angles in these structures (Sup-

porting Information Fig. S4). The variation in main-

chain conformation is high in turns and low in helical

regions, with the notable exception of the HtH region.

Helix E residues 83–89 present almost as much varia-

tion as do turn regions, but residues 80–82 are nearly

as invariant as helical residues of the dimeric core,

with phi, psi angle values near the helical minimum

on the Ramachandran plot. Thus, even though the

wide distribution of HtH conformations in the family

of NMR structures implies apparent instability of this

region, residues 80–82 appear to have intrinsic helical

order. This finding is consistent with the alternative

packing patterns in the crystal structure in which this

helical segment appears to move as a unit.

The seemingly high degree of helical order for

residues 80-82 is also unexpected given their local

sequence environment, as they are embedded within

a segment rich in b-branched and Gly residues:

76-GlyAlaGlyIleAlaThrIleThr-83. This sequence has

relatively low helical propensity (Supporting Infor-

mation Fig. S3). The occurrence of b-branched resi-

dues in helical segments is entropically disfavored

because their side-chains can access only one

rotamer conformation due to steric hindrance

between the bulky Cb branch point and the helical

backbone.36 In principle, the steric hindrance could

be relieved by unraveling of the segment into a more

extended structure, but the resulting excursion of

the chain might compromise tertiary interactions. A

similar argument can be made regarding residues

76-GlyAlaGlyIle-79, which adopt 310-helix conforma-

tion in the distorted L75F apoTrpR subunit. Their

side-chains offer minimal steric hindrance that

would promote extension of the helix rise from a- to

310 conformation, and this sequence also has overall

low helical propensity (Supporting Information Fig.

S3). The fact that it adopts 310 conformation rather

than a-helix or random coil is consistent with a role

for tertiary constraints. Tertiary constraint of sec-

ondary structure was invoked to explain why addi-

tional residues inserted within helical segments of

lysozyme are accommodated not by helix lengthen-

ing but by displacement of adjacent helical residues

into flanking turn regions.37 The coupling of second-

ary and tertiary structures is thought to be the

molecular origin of protein folding cooperativity.38

In TrpR, the tertiary interactions made by the

b-branched residues of helix E or E0 involve Ile57

and Val58, which are within a segment of helix C

that is also rich in b-branched residues, 53-ThrArg-

ValArgIleVal-58. Thus, the distortion and register

shift of the E0 helix act together to accommodate the

en bloc movement of one b-branched segment (79–

83) while maintaining efficient packing with another

b-branched segment (53–58) that preserves its con-

formation. The participation of b-branched residues

in tertiary constraint calls to mind the transmem-

brane helix of glycophorin A,39 in which such residues

play a key role at the quaternary level as well.

b-branched residues are common in helical segments of

membrane proteins, and have been thought to reflect

altered helical propensity in the membrane environ-

ment.40 However, the bilayer acts as a constraint on he-

lix length and on helix–helix packing through its influ-

ence on the exposure of polar and nonpolar residues to

the surrounding lipid, analogous to the tertiary con-

straint in soluble proteins. Helical b-branched residues

present side-chains that are preorganized for interac-

tion with intra- or intermolecular partners, making

them perhaps ideally suited to enforce super-secondary

constraints whether these arise from the tertiary struc-

ture in the case of soluble proteins or the bilayer in the

case of membrane proteins.

Sigler and coworkers had earlier coined the

term ‘‘hydrophobic brace’’ to describe the interactions

between residues in these b-branched-rich segments

because they appeared to fix the intrinsic conforma-

tion of the HtH motif and its orientation relative to

the rest of the molecule in TrpR as well as in several

other HtH repressors.28 In Figure 4, brace residue

positions are indicated by dots. Mutation of Leu75 to

Phe crowds the brace, resulting in rearrangement as

reflected in the alternative packing arrangements

Carey et al. PROTEIN SCIENCE VOL 21:63—74 71

shown in Figure 5. Rearrangement of the brace to

accommodate the mutation implies that the brace is

not a rigid enforcing structure as its name suggests,

but rather is adaptable. The majority of brace resi-

dues in Sigler’s group of repressors are Ile, Leu, and

Val, all of which in addition to being hydrophobic

have severe restrictions on their side-chain confor-

mations. The present analysis thus suggests that a

more complete definition of the brace would high-

light steric constraints on conformational mobility.

Clusters of the branched aliphatic side-chains

Ile, Leu, and Val (ILV) have recently been implicated

as early organizing features of both native and off-

pathway folding intermediates of TIM barrel and

Rossmann fold proteins.41 In native proteins ILV

clusters appear to correlate with regions in which

backbone amides are highly protected from hydrogen

exchange, an effect that has been ascribed to the sol-

vent-excluding property of the branched side-chains

that shields the underlying backbone.42,43 Residues

Val55, Ile57, Val58, and Ile82 from the b-branched

segments of helices C and E, as well as Leu75 from

helix D, belong to the ILV cluster of wt TrpR (S.

Kathuria and C. R. Matthews, personal communica-

tion). However, the backbone amides of both Leu75

and Ile82 are among the most rapidly exchanging in

wt apoTrpR.4 Thus, cluster residue Ile82 is unlikely

to contribute to the apparently intrinsic helical char-

acter of the 80–82 segment of helix E by a solvent-

exclusion mechanism, supporting a role for steric hin-

drance. It is worth noting that even Leu, though not

b-branched, has significant steric restriction that may

contribute to the role of ILV clusters. In the rotamer

library of Dunbrack and Karplus,36 only 2% of the

Leu chi1 conformers lie in the range 60� 6 60�; the

remainder are divided about 2:1 between the other

two conformers, regardless of backbone conformation.

The relative importance of steric hindrance and sol-

vent exclusion in ILV clusters may be resolvable with

a larger database of protein hydrogen-exchange rates.

Conclusions

The crystal structures of isomorphous wt and L75F

apoTrpR that are reported here reveal considerable

perturbation in the L-tryptophan-binding and DNA-

binding HtH region caused by the Leu to Phe substi-

tution. In crystals of the L75F mutant the extent

and nature of structural distortion depend on local

environmental context: an extensive crystal lattice

interaction at one HtH supports a wt-like conforma-

tion, whereas the other HtH lacking crystal contacts

presents a major rearrangement of secondary and

tertiary structure. Given that (i) the lattice interac-

tion supporting the wt-like conformation of L75F

involves an N-terminal arm-HtH contact that is also

likely to be relevant in solution, and (ii) the dis-

torted HtH conformation largely lacks crystal con-

tacts, it is likely that both conformations observed in

the crystal are sampled by L75F apoTrpR in solu-

tion. Inclusion of the these conformers within the

family of accepted NMR structures of L75F apoTrpR

provides a more fully satisfactory explanation of all

existing biophysical and NMR data. The results indi-

cate that distinct alternative conformations can be

overlooked despite full NMR structure determina-

tion as well as detailed relaxation measurements.

Without structural models of specific alternative con-

formers to guide analysis, NMR data may appear to

indicate poor ordering of a segment.

Experimental Procedures

Crystallization and data collectionwt and L75F apoTrpR were purified as described.20,44

Protein stocks were stored frozen at �12 mg/mL in

250 mM NaCl, 0.1 mM PMSF, 0.1 mM sodium phos-

phate, pH 7.0, at �20�C prior to crystallization trials.

Crystals were obtained by standard hanging-drop

vapor diffusion at 294 K with Hampton Research Crys-

tal Screen condition #6 (30% (w/v) PEG 4000, 200 mM

MgCl2, 100 mM Tris HCl pH 8.5). Single crystals grew

overnight with typical dimensions 0.2 mm � 0.2 mm �0.05 mm. One wt apoTrpR crystal and one L75F

apoTrpR crystal were soaked for 30 s in Hampton

Research crystal screen cryo condition #6 (24% (w/v)

PEG 4000, 180 mM MgCl2, 80 mM Tris HCl, pH 8.5,

and 20% (v/v) glycerol), mounted on nylon loops, and

flash-cooled to 100 K in a N2 gas stream. X-ray dif-

fraction data were collected at the Brookhaven

National Laboratory National Synchrotron Light

Source beamline X26C with an ADSC Quantum 4

CCD detector. The incident radiation wavelength was

1.1 A. Reflections were integrated, scaled, and merged

using HKL2000.45 Data collection statistics are pro-

vided in Table I.

Structure determination

Initial phases were obtained for both wt and L75F mu-

tant structures by the molecular replacement method

using the program AMORE.46 In each case the search

model was orthorhombic TrpR dimer2 (PDBid 2oz9),

with L-tryptophan ligands and solvent excluded. Rota-

tion and translation solutions were unambiguous. Each

structure was subjected to multiple cycles of refinement

and manual model-building using Phenix47 and

COOT.48 Atomic displacements were modeled with indi-

vidual isotropic B-factors and a single set of transla-

tion-libration-screw (TLS) parameters for all atoms.

Analysis and displayConformation occupancy test: a composite L75F

apoTrpR model was constructed with residues 74–95

from the HtH2 chain in both wt-like and deformed

conformations, each initially at half occupancy. The

model was subjected to refinement against the X-ray

diffraction data for occupancy only, with no solvent

72 PROTEINSCIENCE.ORG Long-Range Distortion Permitted by Point Mutation

model imposed. The occupancy of the deformed con-

formation increased to 0.95, while the occupancy of

the wt-like conformation decreased to 0.07, indicat-

ing that wt-like conformation is not present in the

HtH2 region of the L75F structure.

The Molprobity server49 was used to evaluate

main-chain conformation, as reported in Table I; the

Molprobity server was also used to add hydrogen

atoms to the crystal structures for comparison of the

L75F apoTrpR crystal structure with NOE restraint

data. Each proton–proton distance was measured and

compared to the cutoff distance assigned on the basis

of the strength of each NOE, taking into account dis-

tance ambiguities for chemically equivalent protons.

L-tryptophan ligand pocket volumes were meas-

ured using the Castp server.50 Because the position

of the Arg84 side-chain varies significantly between

the models compared, the calculations were per-

formed on models in which Arg84 side-chain atoms

beyond Cb were removed.

Secondary structure propensities (Supporting In-

formation Fig. S3) were determined using four differ-

ent propensity scales as follows. Stapley-Doig a: DGvalues for residue in helix relative to residue in coil.51

Stapley-Doig b: DG values for residue in b-strand rela-

tive to Ala in b-strand.51 Minor-Kim b: DDG values for

residue in b-strand relative to Ala in b-strand.52 Pace-

Scholtz a: DDG values for the residue in a-helix rela-

tive to Ala in a-helix.53 For each propensity scale, val-

ues for individual residues were summed and the sum

divided by the number of residues in the segment.

Variance of main-chain torsion angles phi and

psi (Supporting Information Fig. S4) in the 20 L75F

apoTrpR NMR model chains and two X-ray crystal

structure model chains were calculated using Pro-

check-NMR.54

Calculations of accessible surface area (Support-

ing Information Table S1) used the program NAC-

CESS.55 The solvent probe was a water molecule of

1.4 A radius, and the slice size (Z-step) was 0.05 A.

Graphical images were created using Pymol56

(Figs. 1–3, 5, and Supporting Information Fig. S1) and

UCSF Chimera57 (Supporting Information Fig. S2).

Acknowledgments

We thank M. Gryk, G. Rose, and C. Yanofsky for valua-

ble discussions, S. Kathuria and C.R. Matthews for

bringing ILV clusters to our attention and for applying

their analysis to TrpR, G. Montelione for advice on

comparing NOEs to calculated distances, V. Copi�e for

providing the ensemble of models of the L75F apo-trpR

mutant minimized against NMR restraints prior to

their deposition and the NMR relaxation results prior

to publication, and A. Heroux for assistance with data

collection at National Synchrotron Light Source beam-

line X26C, which is supported by the Department of

Energy and the National Institutes of Health (NIH).

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