Sandbox 16: Difference between revisions

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== Alpha-lytic protease ==

'''Alpha-lytic protease (αLP)''' is a 198 residue extracellular bacterial serine protease produced by Lysobacter enzymogenes. The three-dimensional fold of αlp puts it in the same class as cymotrypsin, trypsin and other digestive serine proteases despite only modest sequence homology<ref>PMID:117110</ref>. However, unlike its thermodynamically stable homologs, αLP is stabilized by a large unfolding activation barrier. This kinetic stability optimizes the native state to survive under the harsh, proteolytic conditions in which it operates. Since the native state is less stable than both an intermediate and a completely unfolded state, αLP requires a Pro region to facilitate folding by stabilizing the folding transition state as well as the native state. After folding, the pro region is proteolytically cleaved, leaving an active αLP kinetically trapped

'''Alpha-lytic protease (αLP)''' is a 198 residue extracellular bacterial serine protease produced by Lysobacter enzymogenes. The three-dimensional fold of αlp puts it in the same class as cymotrypsin, trypsin and other digestive serine proteases despite only modest sequence homology<ref>PMID:117110</ref>. However, unlike its thermodynamically stable homologs, αLP is stabilized by a large unfolding activation barrier. This kinetic stability optimizes the native state to survive under the harsh, proteolytic conditions in which it operates. Since the native state is less stable than both an intermediate and a completely unfolded state, αLP requires a Pro region to facilitate folding by stabilizing the folding transition state as well as the native state. After folding, the pro region is proteolytically cleaved, leaving an active αLP kinetically trapped.

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== Structure ==

The <scene name='Sandbox_16/Alp-1/2'>secondary structure</scene> contains the double β-barrel motif (colored yellow) that is characteristic of the chymotrypsin family as well as an active site containing the <scene name='Sandbox_16/Alp-1/6'>"catalytic triad"</scene> - His 57, Asp 102, and Ser 195 - that is responsible for proteolysis. The preference for αLP to cleave substrates on the C-terminal side of small hydrophobic residues, such as Alanine and Valine is mostly due to <scene name='Sandbox_16/Alp-1/3'>three residues in the S1 pocket</scene> consisting of Met 190, Met 213, and Val 218<ref>PMID:9232638</ref>. The N to C coloring and important structural regions are shown <scene name='Sandbox_16/Alp-1/8'>here</scene>, ~~where~~ the molecule is colored dark blue at the N-terminus progressing to red at the C-terminus. In contrast to its mammalian homologs like trypsin and chymotrypsin, αLP is synthesized with a 166 residue N-terminal Pro region that plays an obligatory role in the proper folding of its 198 residue protease domain<ref>PMID:2646278</ref>. The Pro region overcomes the barrier to folding by providing a catalyzed pathway in which the transition state to folding is lowered by 18.2 kcal/mol<ref>PMID:9796818</ref>. The product of this folding is not active αLP but an inhibitory complex, N*P. The release of active αLP requires the removal of the Pro region via proteolysis, which occurs naturally. This leaves the native αLP, a metastable state with a large barrier to unfolding (t1/2~1.2 years). Below shows the free energy diagram summarizing the difference between the folding landscape of a typical thermodynamically stable protein (left) and that of αLP (right). The dotted blue line indicates the folding landscape in the presence of the Pro region.

The <scene name='Sandbox_16/Alp-1/2'>secondary structure</scene> contains the double β-barrel motif (colored yellow) that is characteristic of the chymotrypsin family as well as an active site containing the <scene name='Sandbox_16/Alp-1/6'>"catalytic triad"</scene> - His 57, Asp 102, and Ser 195 - that is responsible for proteolysis. The preference for αLP to cleave substrates on the C-terminal side of small hydrophobic residues, such as Alanine and Valine is mostly due to <scene name='Sandbox_16/Alp-1/3'>three residues in the S1 pocket</scene> consisting of Met 190, Met 213, and Val 218<ref>PMID:9232638</ref>. The N to C coloring and important structural regions are shown <scene name='Sandbox_16/Alp-1/8'>here</scene>, with the molecule colored dark blue at the N-terminus and progressing to red at the C-terminus. The Domain Bridge is the only covalent linkage between the two domains and has been shown to modulate the unfolding rate<ref>PMID:17543987</ref>. In contrast to its mammalian homologs like trypsin and chymotrypsin, αLP is synthesized with a 166 residue N-terminal Pro region that plays an obligatory role in the proper folding of its 198 residue protease domain<ref>PMID:2646278</ref>. The Pro region overcomes the barrier to folding by providing a catalyzed pathway in which the transition state to folding is lowered by 18.2 kcal/mol<ref>PMID:9796818</ref>. The product of this folding is not active αLP but an inhibitory complex, N*P. The release of active αLP requires the removal of the Pro region via proteolysis, which occurs naturally. This leaves the native αLP, a metastable state with a large barrier to unfolding (t1/2~1.2 years). Below shows the free energy diagram summarizing the difference between the folding landscape of a typical thermodynamically stable protein (left) and that of αLP (right). The dotted blue line indicates the folding landscape in the presence of the Pro region.

[[Image:therm vs kinetic.jpg]]

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@@ Line 1: / Line 1: @@
 == <p style="font-size:x-large;">Alpha-lytic protease</p> ==
-'''Alpha-lytic protease (αLP)''' is a 198 residue extracellular bacterial serine protease produced by Lysobacter enzymogenes. The three-dimensional fold of αlp puts it in the same class as cymotrypsin, trypsin and other digestive serine proteases despite only modest sequence homology<ref>PMID:117110</ref>. However, unlike its thermodynamically stable homologs, αLP is stabilized by a large unfolding activation barrier. This kinetic stability optimizes the native state to survive under the harsh, proteolytic conditions in which it operates. Since the native state is less stable than both an intermediate and a completely unfolded state, αLP requires a Pro region to facilitate folding by stabilizing the folding transition state as well as the native state. After folding, the pro region is proteolytically cleaved, leaving an active αLP kinetically trapped
+'''Alpha-lytic protease (αLP)''' is a 198 residue extracellular bacterial serine protease produced by Lysobacter enzymogenes. The three-dimensional fold of αlp puts it in the same class as cymotrypsin, trypsin and other digestive serine proteases despite only modest sequence homology<ref>PMID:117110</ref>. However, unlike its thermodynamically stable homologs, αLP is stabilized by a large unfolding activation barrier. This kinetic stability optimizes the native state to survive under the harsh, proteolytic conditions in which it operates. Since the native state is less stable than both an intermediate and a completely unfolded state, αLP requires a Pro region to facilitate folding by stabilizing the folding transition state as well as the native state. After folding, the pro region is proteolytically cleaved, leaving an active αLP kinetically trapped.
 {{STRUCTURE_2alp |  PDB=2alp  |  SCENE=  }}
@@ Line 6: / Line 6: @@
 == Structure ==
-<p>The <scene name='Sandbox_16/Alp-1/2'>secondary structure</scene> contains the double β-barrel motif (colored yellow) that is characteristic of the chymotrypsin family as well as an active site containing the <scene name='Sandbox_16/Alp-1/6'>"catalytic triad"</scene> -  His 57, Asp 102, and Ser 195 - that is responsible for proteolysis. The preference for αLP to cleave substrates on the C-terminal side of small hydrophobic residues, such as Alanine and Valine is mostly due to <scene name='Sandbox_16/Alp-1/3'>three residues in  the S1 pocket</scene> consisting of Met 190, Met 213, and Val 218<ref>PMID:9232638</ref>. The N to C coloring and important structural regions are shown <scene name='Sandbox_16/Alp-1/8'>here</scene>, where the molecule is colored dark blue at the N-terminus progressing to red at the C-terminus. In contrast to its mammalian homologs like trypsin and chymotrypsin, αLP is synthesized with a 166 residue N-terminal Pro region that plays an obligatory role in the proper folding of its 198 residue protease domain<ref>PMID:2646278</ref>. The Pro region overcomes the barrier to folding by providing a catalyzed pathway in which the transition state to folding is lowered by 18.2 kcal/mol<ref>PMID:9796818</ref>. The product of this folding is not active αLP but an inhibitory complex, N*P. The release of active αLP requires the removal of the Pro region via proteolysis, which occurs naturally. This leaves the native αLP, a metastable state with a large barrier to unfolding (t<sub>1/2</sub>~1.2 years). Below shows the free energy diagram summarizing the difference between the folding landscape of a typical thermodynamically stable protein (left) and that of αLP (right). The dotted blue line indicates the folding landscape in the presence of the Pro region.
+<p>The <scene name='Sandbox_16/Alp-1/2'>secondary structure</scene> contains the double β-barrel motif (colored yellow) that is characteristic of the chymotrypsin family as well as an active site containing the <scene name='Sandbox_16/Alp-1/6'>"catalytic triad"</scene> -  His 57, Asp 102, and Ser 195 - that is responsible for proteolysis. The preference for αLP to cleave substrates on the C-terminal side of small hydrophobic residues, such as Alanine and Valine is mostly due to <scene name='Sandbox_16/Alp-1/3'>three residues in  the S1 pocket</scene> consisting of Met 190, Met 213, and Val 218<ref>PMID:9232638</ref>. The N to C coloring and important structural regions are shown <scene name='Sandbox_16/Alp-1/8'>here</scene>, with the molecule colored dark blue at the N-terminus and progressing to red at the C-terminus. The Domain Bridge is the only covalent linkage between the two domains and has been shown to modulate the unfolding rate<ref>PMID:17543987</ref>. In contrast to its mammalian homologs like trypsin and chymotrypsin, αLP is synthesized with a 166 residue N-terminal Pro region that plays an obligatory role in the proper folding of its 198 residue protease domain<ref>PMID:2646278</ref>. The Pro region overcomes the barrier to folding by providing a catalyzed pathway in which the transition state to folding is lowered by 18.2 kcal/mol<ref>PMID:9796818</ref>. The product of this folding is not active αLP but an inhibitory complex, N*P. The release of active αLP requires the removal of the Pro region via proteolysis, which occurs naturally. This leaves the native αLP, a metastable state with a large barrier to unfolding (t<sub>1/2</sub>~1.2 years). Below shows the free energy diagram summarizing the difference between the folding landscape of a typical thermodynamically stable protein (left) and that of αLP (right). The dotted blue line indicates the folding landscape in the presence of the Pro region.
 [[Image:therm vs kinetic.jpg]]