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Coupled vibrations in peptides and proteins: Structural information using 2D-IRspectroscopy

Huerta Viga, A.

Publication date2014

Link to publication

Citation for published version (APA):Huerta Viga, A. (2014). Coupled vibrations in peptides and proteins: Structural informationusing 2D-IR spectroscopy.

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Download date:20 Jul 2021

7How Does Guanidinium

Denature Proteins?

Guanidinium (Gdm+) is a widely used denaturant, but it is still largely unknownhow it operates at the molecular level. In particular, the effect of Gdm+ on the dif-ferent types of secondary structure in proteins is at present not clear. Here we usetwo-dimensional infrared spectroscopy (2D-IR) spectroscopy to investigate changesin the secondary structure of two proteins with mainly α-helical or β-sheet contentupon addition of Gdm-13C15N3·Cl. We find that upon Gdm+-denaturation, the β-sheet protein shows a complete loss of β-sheet structure, whereas the α-helical pro-tein maintains most of its secondary structure. These results suggest that Gdm+ dis-rupts β-sheets much more efficiently than α-helices, possibly because in the formerhydrophobic interactions are more important and the number of dangling hydrogenbonds is larger.

7.1 Introduction

Significant effort has been dedicated to elucidating the molecular mechanism of theeffect of chemical denaturants (reagents that decrease protein stability when addedto the aqueous solvent), the most effective one of which is the guanidinium cation(Gdm+).1,107,108 There are two mechanisms by which a denaturant could destabi-lize proteins: indirectly, by altering the solvent properties of water, and directly,

63

7. How Does Guanidinium Denature Proteins?

by specifically interacting with groups of the protein. The indirect mechanism ismost likely not applicable for Gdm+, since there is ample experimental evidencethat the interaction between water molecules and Gdm+ is very weak.109,110 How-ever, Gdm+ does seem to affect the water-water interactions in the hydration shellof other solutes,111 and it has been proposed that the lack of a hydration shell ofGdm+ promotes solubility of non-polar groups in the interior of proteins.109 Hence,Gdm+ most likely denatures proteins by interacting directly with them, but not withthe backbone because it has been shown that Gdm+ does not form hydrogen bondswith peptide groups.112 The denaturation mechanism of Gdm+ therefore involvesinteractions with the side chains of proteins, but the physical nature of these interac-tions is only just starting to become clear.112–116 At present it is not known whetherGdm+ disrupts certain types of protein-secondary structure more efficiently thanothers, as most studies to date focus on interactions between Gdm+ and the sidechains of proteins.112–116 Here, we study the Gdm+ denaturation of two proteins bydirectly probing their secondary structure using two-dimensional infrared (2D-IR)spectroscopy. Studying Gdm+ denaturation of proteins with IR is difficult becauseGdm+ absorbs at 1600 cm−1 due to a mode involving a combined CN3 antisym-metric stretch and NH2 scissors motion.117 This band overlaps with the Amide I′

band and overwhelms it at the Gdm+ concentrations needed to denature proteins.Although it is in principle possible to subtract this contribution,118 the conditionsunder which this subtraction is possible are limited. To avoid this problem we usea guanidinium isotope, Gdm-13C15N3. The absorption frequency of this isotope isred-shifted by about 60 cm−1 (see Figure 9.4), leaving a clear spectral window aroundthe Amide I′ frequency.

Figure 7.1: (A) Structure of lysozyme (PDB-ID 2LYZ, 129 residues), which contains 41% helical- and 10%β-sheet-secondary structure. (B) Structure of α-chymotrypsin (PDB-ID 1YPH, 241 residues), which contains11% helical- and 34% β-sheet-secondary structure. β sheets are shown in green, α helices in red, 310helices in pink and random coils in gray.

We investigate two well-known proteins: lysozyme , which is mainly α-helical, andα-chymotrypsin, which is mostly formed by β-sheets (see Figure 7.1A and B, respec-tively). Both proteins undergo an unfolding reaction upon addition of Gdm·Cl.119,120

64

7.2. Lysozyme

The native-state 2D-IR spectra of these proteins have been determined before,78,121

and our spectra are in agreement with these earlier measurements. Here we focuson the changes in the 2D-IR spectra of these proteins upon denaturation by Gdm+

by measuring a series of 2D-IR spectra for increasing concentration of Gdm+ aroundthe transition point of the unfolding reaction.

7.2 Lysozyme

The FTIR spectra of native and denatured lysozyme (5 M Gdm+ concentration) areshown in Figure 7.2A and U, respectively. The Amide I′ band of denatured lyso-zyme is blue shifted with respect to the spectra of the native protein, indicating andincreased random-coil content. Figure 7.2C shows the 2D-IR spectrum of folded ly-sozyme for perpendicular polarization of pump and probe pulses, and in Figure 7.2Dwe show the 2D-IR polarization-difference spectrum (3∆α⊥−∆α‖, which eliminatesthe diagonal peaks and leaves only cross peaks),26 in which the coupled A and E1modes of the α-helix are visible. The frequency splitting between these modes isconsistent with theoretical predictions,10 and previous 2D-IR measurements on α-helices.122 Figure 7.2W shows the 2D-IR spectrum of Gdm+-denatured lysozyme.In Figure 7.3A we show slices through the diagonal of the parallel-polarization 2D-IR spectra at increasing Gdm+ concentrations, where the blue shift of the Amide I′

band is clear, confirming the larger amplitude at the random-coil frequency. Sur-prisingly, the difference-polarization spectrum, shown in Figure 7.2X, remains es-sentially the same as that of folded lysozyme. Figure 7.3B shows cross sections alongthe dashed line in these difference spectra for increasing concentration of Gdm+. Lit-tle change in the amplitude of the cross peak is observed upon addition of Gdm+.In the inset of Figure 7.3B, we plot the amplitude of this cross peak (maximum ofthe positive part minus the minimum of the negative one, normalized to the valueof folded lysozyme) as a function of Gdm+ concentration, and it can be seen that itremains roughly constant. This result demonstrates that α-helical structure is pre-served upon denaturation of the protein by Gdm+. Lysozyme has four disulfidebonds that stabilize the globular structure of the protein,123 and Gdm+ will not dis-rupt these tertiary contacts. Therefore, the increase in random-coil conformation thatwe observe is probably due to loss of the 10% beta-sheet structure and of the turns asthe protein unfolds and looses part of its tertiary structure. This interpretation agreeswith optical-rotation studies which showed that lysozyme still has a certain degreeof intra-molecular order and thus is not completely a random coil at 5 M concentra-tion of Gdm+, although the denaturation reaction is completed.119 Furthermore, itwas shown recently that at this same Gdm+ concentration lysozyme has about 70%of its enzymatic activity.124 This high percentage of activity suggest that the remain-ing α-helical structure of lysozyme, partly kept together by the disulfide bonds, isresponsible for the activity of the protein.

65

7. How Does Guanidinium Denature Proteins?

Figure7.2:FT

IRand

2D-IR

spectraoflysozym

eatincreasing

Gdm

+concentrations.

(A)FTIR

spectrumofnative-state

lysozyme.

(B)and(C

)2D-IR

spectrafor

parallelandperpendicular

polarizationof

pump

andprobe

pulses;thecontour

intervalsare

0.4and

0.13m

O.D

,respectively.(D

)D

ifference2D

-IRspectrum

(3∆α⊥

−∆α‖ );the

contourintervals

are0.15

mO

.D.(E)-(H

)Spectrafor

1M

Gdm

+concentration;contour

intervalsare

0.5,0.17and

0.15m

O.D

.(I)-(L)Spectrafor

2M

Gdm

+concentration;contour

intervalsare

0.5,0.17and

0.15m

O.D

.(M)-(P)

Spectrafor

3M

Gdm

+concentration;contour

intervalsare

0.5,0.15and

0.15m

O.D

.(Q)-(T)

Spectrafor

4M

Gdm

+concentration;contour

intervalsare

0.6,0.2and

0.15m

O.D

.(U)-(X

)Spectra

for5

MG

dm+

concentration;contourintervals

are0.8,0.26

and0.15

mO

.D.

66

7.3. α-Chymotrypsin

Figure 7.3: (A) Diagonal of the 2D-IR spectra of lysozyme at increasing Gdm+ concentrations. (B) Crosssections of the difference 2D-IR spectra of lysozyme for νpump = 1672 cm−1, at increasing Gdm+ concen-trations. In the inset the fraction of α-helical content of lysozyme as a function of Gdm+ concentration isshown (the line is a guide to the eye).

7.3 α-Chymotrypsin

α-Chymotrypsin shows a different behavior upon addition of Gdm+. This proteincompletes a denaturation reaction at 4 M Gdm+ concentration.119 Its structure isshown in Figure 7.1B. The FTIR spectra of native and denatured α-chymotrypsin(4 M Gdm+ concentration) are shown in Figures 7.4A and Q, respectively. Just asfor lysozyme, the Amide I′ band of native and denatured α-chymotrypsin under-goes a blue shift, indicating increased random-coil conformation. The perpendicular-polarization 2D-IR spectrum of the native state of the protein, shown in Figure 7.4C,is dominated by the two coupled Amide I′ modes of the β-sheet structure at 1620and 1675 cm−1.78 These coupled modes give rise to a 2D-IR line shape that is typicalof β-sheets, due to the presence of strong cross peaks. The polarization difference2D-IR spectrum, shown in Figure 7.4D, shows the isolated cross-peaks between theβ-sheet modes, as well as a small α-helical contribution. At a Gdm+ concentrationof 4 M the 2D-IR spectrum (shown in Figure 7.4R-T) has lost this typical β-sheet lineshape. The parallel and perpendicular polarization spectra resemble more those ofa random-coil conformation, which is more easily seen in the diagonal slices at in-creasing Gdm+ concentrations, shown in Figure 7.5A. The polarization difference2D-IR spectrum of the denatured protein only shows traces of structure, probablyarising from its small α-helical content. In Figure 7.5B we show cross-sections of theperpendicular-polarization 2D-IR spectra for a pump frequency resonant with thehigh-frequency β-sheet mode (1688 cm−1), for increasing concentrations of Gdm+.These cross-sections show that both the diagonal peak and corresponding cross peakdecrease significantly at moderate Gdm+ concentrations, and have completely van-ished at a concentration of 4 M. In the inset of this figure we plot the amplitudeof the β-sheet cross peak as a function of Gdm+ concentration, and a clear decayto zero is observed. This result indicates that the β-sheet secondary structure of α-chymotrypsin is completely destabilized by the presence of the Gdm+ ions. This

67

7. How Does Guanidinium Denature Proteins?

Figure7.4:FT

IRand

2D-IR

spectraofα

-chymotrypsin

atincreasingG

dm+

concentrations.(A)FTIR

spectrumofnative-state

α-chym

otrypsin.(B)and(C

)2D-IR

spectrafor

parallelandperpendicular

polarizationof

pump

andprobe

pulses;thecontour

intervalsare

0.8and

0.3m

O.D

,respectively.(D

)D

ifference2D

-IRspectrum

(3∆α⊥

−∆α‖ );the

contourintervals

are0.2

mO

.D.(E)-(H

)Spectra

for1

MG

dm+

concentration;contourintervals

are0.5,0.18

and0.2

mO

.D.(I)-(L)

Spectrafor

2M

Gdm

+concentration;contour

intervalsare

0.25,0.13and

0.2m

O.D

.(M)-(P)Spectra

for3

MG

dm+

concentration;contourintervals

are0.4,0.15

and0.2

mO

.D.(Q

)-(T)Spectrafor

4M

Gdm

+concentration;contour

intervalsare

0.3,0.1and

0.2m

O.D

.

68

7.4. Discussion

finding is consistent with the complete loss of enzymatic activity of α-chymotrypsin,which occurs at a concentration of 3 M.125

Figure 7.5: (A) Diagonal slices of the 2D-IR spectra of α-chymotrypsin at increasing Gdm+ concentrations.(B) Cross sections of the perpendicular 2D-IR spectra of α-chymotrypsin for νpump = 1688 cm−1. In theinset the fraction of β-sheet content of α-chymotrypsin as a function of Gdm+ concentration is shown (theline is a guide to the eye).

7.4 Discussion

Our results strongly suggest that Gdm+ efficiently disrupts the tertiary structure ofproteins as well as their β-sheet secondary structure, but does not significantly desta-bilize α-helical secondary structure. It should be noted that the increased sensitivityof 2D-IR spectroscopy (as compared to conventional IR spectroscopy) to secondaryprotein structure was essential in reaching this conclusion. Our observations couldbe explained by a denaturation mechanism that would involve a strong decreaseof the hydrophobic effect via direct interactions of Gdm+ with the side chains ofthe proteins, thus promoting the exposure of hydrophobic-core residues. α-helicesare often amphipatic,12 and Gdm+ seems to promote solvation of their hydrophobicside, disrupting in this manner the tertiary structure of the protein, but leaving in-tact the α-helices themselves, probably because these are generally not significantly

Figure 7.6: Illustration of the proposed mechanism of protein denaturation with guanidinium: β-sheetsvanish at low Gdm+ concentrations and α-helices remain as the protein extends and looses its tertiarystructure.

69

7. How Does Guanidinium Denature Proteins?

stabilized by hydrophobic interactions, but rather by the intrinsic helical propensityof specific amino acids.11 On the contrary, hydrophobic interactions between sidechains promote the formation of β-sheets,12 and we find that Gdm+ destabilizesthem very efficiently, most likely because it promotes solvation of their non-polargroups, thus facilitating the detachment of the β-strands from each other. In addi-tion to the hydrophobic effect, the difference in hydrogen bonding topology betweenα-helices and β-sheets could make α-helices more difficult to chemically denaturethan β-sheets. In α-helices each C=O group of residue i forms a hydrogen bondwith the N-H of residue i + 4, except the first N-H groups and the final four C=Ogroups which lack a hydrogen bond within the helix. However, it was found thatnumerous naturally-occurring helices meet the condition of having flanking residueswhose side chains can form these missing intra-helical hydrogen bonds,126 resultingin more stable helices. This means that the majority of the residues in α-helices formhelix-stabilizing hydrogen bonds. On the other hand, in β-sheets hydrogen bondsare formed between neighboring β-strands, therefore one half of the edge strandsof β-sheets is not involved in intra-sheet hydrogen-bond interactions. Hence, for asheet consisting of two strands, only 50% of the residues will be forming hydrogenbonds that stabilize the β-sheet. For β-sheets with three strands, like most of theones that constitute α-chymotrypsin, the number of participating residues increasesto 60%, which is still much less than in α-helices. This difference could partly accountfor the β-sheets of α-chymotrypsin being disrupted at low Gdm+ concentrations, asfewer hydrogen bonds keep the β-strands together after Gdm+ has promoted sol-vation of the non-polar side chains. However, for a larger number of β-strands, thepercentage of hydrogen-bonding residues increases, which might explain why largerβ-sheets constructs, like amyloid fibrils, are more difficult to chemically denaturewith Gdm+.127

7.5 Conclusion

As illustrated in Figure 7.6, we find evidence that α-helical structure remains inproteins after denaturation with Gdm+, whereas β-sheets are easily destabilized.Residual structure in chemically denatured proteins is a controversial topic, espe-cially because experimental probes of secondary structure in such conditions arelimited. However, current experimental evidence indicates that the remaining struc-ture (if any) is sequence local rather than long range.128 Our study shows that 2D-IRspectroscopy provides a robust way of overcoming the challenges of investigatingsecondary structure in chemically denatured proteins, and our findings are in agree-ment with the existence of residual structure (either native or non-native) of local-sequence character.

70