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2. Several techniques were used to study the degree of translocation of truncated intermediates in the synthesis of an integral membrane protein (with a single transmembrane domain). Experiments were performed in the presence of SRP and canine pancreatic microsomes. The intermediates were synthesized using a rabbit reticulocyte translation system in the presence of 35S-methionine. Of the techniques used to monitor the progress of synthesis and translocation/insertion,| we are concerned with the following: I Sedimentation: chains associated with the microsomes are found in the pellet of a 75,000 rpm ultracentrifuge spin. •Crosslinks: a photoreactive phenylalanine derivative was incorporated into the truncated chain, and crosslinking to protein and lipid was analyzed after photo-activation. The intermediates studied using these approaches had between 40 and 154 amino acids. All truncated proteins with more than 60 amino acids sediment by centrifugation at neutral pH, while only intermediates with more than 71 amino acids sediment at alkaline pH (pH 9.5). Chains with between 60 and 71 amino acids are sedimented at neutral but not alkaline pH. With the knowledge that alkaline pH can disrupt protein-protein interactions, but not protein-lipid interactions, explain the sedimentation characteristics of the truncated intermediates in the context of their translocation state (i.e. where are the intermediates in the process of being integrated into the membrane?).

2. Several techniques were used to study the degree of translocation of truncated intermediates in the synthesis of an integral membrane protein (with a single transmembrane domain). Experiments were performed in the presence of SRP and canine pancreatic microsomes. The intermediates were synthesized using a rabbit reticulocyte translation system in the presence of 35S-methionine. Of the techniques used to monitor the progress of synthesis and translocation/insertion,| we are concerned with the following: I Sedimentation: chains associated with the microsomes are found in the pellet of a 75,000 rpm ultracentrifuge spin. •Crosslinks: a photoreactive phenylalanine derivative was incorporated into the truncated chain, and crosslinking to protein and lipid was analyzed after photo-activation. The intermediates studied using these approaches had between 40 and 154 amino acids. All truncated proteins with more than 60 amino acids sediment by centrifugation at neutral pH, while only intermediates with more than 71 amino acids sediment at alkaline pH (pH 9.5). Chains with between 60 and 71 amino acids are sedimented at neutral but not alkaline pH. With the knowledge that alkaline pH can disrupt protein-protein interactions, but not protein-lipid interactions, explain the sedimentation characteristics of the truncated intermediates in the context of their translocation state (i.e. where are the intermediates in the process of being integrated into the membrane?).

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
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2. Several techniques were used to study the degree of translocation of truncated intermediates in the synthesis of an integral membrane protein (with a single transmembrane domain). Experiments were performed in the presence of SRP and canine pancreatic microsomes. The intermediates were synthesized using a rabbit reticulocyte translation system in the presence of 35S-methionine. Of the techniques used to monitor the progress of synthesis and translocation/insertion,| we are concerned with the following: Sedimentation: chains associated with the microsomes are found in the pellet of a 75,000 rpm ultracentrifuge spin. Crosslinks: a photoreactive phenylalanine derivative was incorporated into the truncated chain, and crosslinking to protein and lipid was analyzed after photo-activation. The intermediates studied using these approaches had between 40 and 154 amino acids. All truncated proteins with more than 60 amino acids sediment by centrifugation at neutral pH, while only intermediates with more than 71 amino acids sediment at alkaline pH (pH 9.5). Chains with between 60 and 71 amino acids are sedimented at neutral but not alkaline pH. With the knowledge that alkaline pH can disrupt protein-protein interactions, but not protein-lipid interactions, explain the sedimentation characteristics of the truncated intermediates in the context of their translocation state (i.e. where are the intermediates in the process of being integrated into the membrane?). C
Transcribed Image Text:2. Several techniques were used to study the degree of translocation of truncated intermediates in the synthesis of an integral membrane protein (with a single transmembrane domain). Experiments were performed in the presence of SRP and canine pancreatic microsomes. The intermediates were synthesized using a rabbit reticulocyte translation system in the presence of 35S-methionine. Of the techniques used to monitor the progress of synthesis and translocation/insertion,| we are concerned with the following: Sedimentation: chains associated with the microsomes are found in the pellet of a 75,000 rpm ultracentrifuge spin. Crosslinks: a photoreactive phenylalanine derivative was incorporated into the truncated chain, and crosslinking to protein and lipid was analyzed after photo-activation. The intermediates studied using these approaches had between 40 and 154 amino acids. All truncated proteins with more than 60 amino acids sediment by centrifugation at neutral pH, while only intermediates with more than 71 amino acids sediment at alkaline pH (pH 9.5). Chains with between 60 and 71 amino acids are sedimented at neutral but not alkaline pH. With the knowledge that alkaline pH can disrupt protein-protein interactions, but not protein-lipid interactions, explain the sedimentation characteristics of the truncated intermediates in the context of their translocation state (i.e. where are the intermediates in the process of being integrated into the membrane?). C
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