Chymotrypsin inhibitor 2 (CI2) is a monomeric protein of 64 residues and folds by simple two‐state kinetics. The only well developed region during intermediate state is an N‐terminal α‐helix and some distant residues in sequence which contact with it. The α‐helix packs with the β‐sheet to form the hydrophobic core. Studies31,32 showed that the slowest exchange‐rate residues are located in the C‐terminus of the α‐helix (I20‐I21) and the central strand of β‐sheet (V47, L49‐V51); the other slowest exchange amide protons are K11(β2), I30 and L32 (β3).
Figure 1.6 Conformational entropy profile of chymotrypsin inhibitor 2. Residues with the slowest exchanging protons (blue) and the lowest conformational entropy (red) are labeled.
Figure 1.6 shows that most residues having the lowest conformational entropy overlap with those of the slowest hydrogen exchange rates, except K11. Most of them are on the α‐helix, β‐strands 3 and 4. Figure 1.7 compares the spatial distribution of the lowest conformational entropy and the slowest exchange‐rate regions on ribbon diagram of CI2.
Figure 1.7 Spatial distribution of residues with the slowest exchanging protons (blue) and the lowest conformational entropy (red) on the ribbon diagram of chymotrypsin inhibitor 2.
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Cytochrome c
Hydrogen exchange study33 shows that the slowest exchanging amide protons are located in the three major helical segments of cytochrome c. F10 (N‐helix) and L94‐K99 (C‐helix) carry the slowest exchanging amide protons.
Figure 1.8 Conformational entropy profile of cytochrome c. Residues with the slowest exchanging protons (blue) and the lowest conformational entropy (red) are labeled.
Figure 1.8 shows the conformational entropy profile of cytochrome c.
I9‐V11 and D93‐L98 are the residues having the lowest entropy values which match the slowest exchanging residues on N‐helix and C‐helix suggested by experiment. Note that the 60’s helix also has relative low entropy values. This is consistent with the experimental results that the next slowest exchanging amide protons are located in the 60’s helix. Figure 1.9 shows the spatial distribution of residues with the slowest exchanging protons and the lowest conformational entropy on ribbon diagram of cytochrome c.
Figure 1.9 Spatial distribution of residues with the slowest exchanging protons (blue) and the lowest conformational entropy (red) on the ribbon diagram of cytochrome c.
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Equilibrium Protein Folding
Cytochrome c is a good model system in studies34‐36 of protein folding and unfolding. Under native conditions, most proteins exist in their unique native conformation. However some of them also exist in higher energy states and continue to cycle through totally unfolded states and partially folded states. These non‐native forms are usually hard to be detected because of the abundant native conformational signals. Hydrogen exchange experiment can be used to detect these partially folded conformations and define the unfolding units of proteins.
Through HX studies34‐36, there are four cooperative unfolding units defined in cytochrome c: the blue bi‐helix (B), the green Ω loop and the 60’s helix (G) , the yellow (Y) and the red Ω loop (R) in the order of decreasing unfolding free energy. These unfolding units may define the folding and unfolding pathways of cytochrome c by forming various intermediates through different combinations.
B G Y G R B
Figure 1.10 Conformational entropy profile of each unfolding unit of cytochrome c and the corresponding average values.
Figure 1.10 shows the conformational entropy profile of each unfolding unit and the corresponding average values. Unit B has the lowest average conformational entropy, unit G has second lowest entropy, and then Y, and R.
The order of increasing conformational entropy follows the order of decreasing unfolding free energy.
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Barnase
Barnase has three α‐helices and a five‐stranded β‐sheet. The first helix packs onto the β‐sheet to form the major hydrophobic core of the protein. The second and the third α‐helices pack onto another side of the β‐sheet to form a smaller hydrophobic core.
Figure 1.11 Conformational entropy profile of barnase. Residues with the slowest exchanging protons (blue) and the lowest conformational entropy (red) are labeled.
Figure 1.11 shows the conformational entropy profile of barnase.
Previous study37,38 showed that L14, I25, A74, L89 and Y97 have the lowest exchange rates. L14 on the first α‐helix and I25, A74 ad L89 on the β‐sheet are located in the major hydrophobic core of the protein. Y97 is in the center of the smaller hydrophobic core formed by the two smaller helices and part of the β‐sheet. Figure 1.12 shows the spatial distribution of residues with the slowest exchanging protons and the lowest conformational entropy on the ribbon diagram of barnase.
Figure 1.12 Spatial distribution of residues with the slowest exchanging protons (blue) and the lowest conformational entropy (red) on the ribbon diagram of barnase.
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α‐Lactalbumin
α‐lactalbumin is a calcium‐binding protein which consists of a β‐sheet domain and a α‐helical domain. The helical domain is composed of four helices: helix A (C6‐E11), helix B (P24‐S34), helix C (D87‐D97) and helix D (D102‐ L105). Previous studies39,40 showed that in the helical domain, the C‐helix is the most protected, having the lowest exchanging rate, followed by the B and then the A‐helix.
Figure 1.13 Conformational entropy profile of α‐lactalbumin. Residues with the slowest exchanging protons (blue) and the lowest conformational entropy (red) are labeled.
Figure 1.13 shows the profile of the conformational entropy. The residues in C‐helix have the lowest conformational entropy and the residues which have second lowest conformational entropy are located in helix B and helix A.
The regions which have low conformational entropy are consistent with those having slow exchanging rate39,40. Note that a previous study40 showed that helix D exchanges too fast and the exchanging rates are not measurable. Our calculation also indicates that helix D has high conformational entropy. Figure 1.14 shows the spatial distribution of residues with the slowest exchanging
protons and the lowest conformational entropy on the ribbon diagram of α‐lactalbumin.
Figure 1.14 Spatial distribution of residues with the slowest exchanging protons (blue) and the lowest conformational entropy (red) on the ribbon diagram of α‐lactalbumin.
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Cardiotoxin III
Cardiotoxin III (CTX III) is a 60‐amino acid, all β‐sheet protein. The secondary structure of CTX III includes five β‐strands forming double and triple‐stranded anti‐parallel β‐sheets. Hydrogen exchange study41 on CTX III showed that residues K23, I39, and Y51‐N55 constitute the hydrophobic cluster of the protein.
Figure 1.15 Conformational entropy profile of CTX III. Residues with the slowest exchanging rotons (blue) and the lowest conformational entropy (red) are labeled.
d Figure spatial
the lowest conformational entropy on the ribbon diagram of cardiotoxin III.
p
Figure 1.15 shows the conformational entropy profile of CTX III. Our calculation of low conformational entropy residues covers most of the slow exchange resi ues (Y22‐M24, V52). 1.16 shows the distribution of residues with the slowest exchanging protons and
Figure 1.16 Spatial distribution of residues with the slowest exchanging protons (blue) and the lowest conformational entropy (red) on the ribbon diagram of CTX III.
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Ribonuclease H
Ribonuclease H (RNaseH) is a 155‐residue protein with four α‐helices packing with a five‐stranded β‐sheet. A previous study42 showed that helix A (T43‐L56) and helix D (V101‐L111) are the most stable regions in the protein.
Figure 1.17 Conformational entropy profile of ribonuclease H. Residues with the slowest exchanging protons (blue) and the lowest conformational entropy (red) are labeled.
Figure 1.17 shows the conformational entropy profile of RNaseH. The slowest exchanging residues are L49‐I53 and A55‐L56 which are located on helix A. The conformational entropies of L49‐V54 are the lowest from our calculation. L107 on helix D also has slow exchanging rate and its conformational entropy is relative low. Figure 1.18 shows the spatial distribution of residues with the slowest exchanging protons and the lowest conformational entropy on the ribbon diagram of RNaseH.
Figure 1.18 Spatial distribution of residues with the slowest exchanging protons (blue) and the lowest conformational entropy (red) on the ribbon diagram of
ribonuclease H.
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