Part C-June 2014

CSIR-UGC National Eligibility Test (NET) for Junior Research Fellowship and Lecturer-ship

Part C


This time CSIR does not allow candidates to carry questions with them. We have collected maximum questions from our candidates (memory based).

1. You are studying a protein that inserts itself into a model membrane (liposomes) during a reconstitution process. The protein has an N-terminal, 18-amino acid hydrophilic segment that is located on the outside of the membrane, a 19-amino acid hydrophobic transmembrane segment flanked by negatively and positively charged amino acids, and a C-terminal domain that resides inside the lumen (as depicted below in the form of a cartoon)

For proper reconstitution of the protein, which of the following strategies will be appropriate ?

1.   Increase the number of negatively charged amino acids in the N-terminal
2.   Increase the number of positively charged amino acids in the C-terminal
3.   Removal of positively charged amino acids from the C-terminal
4.   Increase the length of hydrophobic segment

Ans: 4
Expln:- Membrane proteins are classified as either peripheral (extrinsic) or integral (intrinsic) depending on how tightly they are associated with the membrane. Peripheral proteins are less tightly bound to the lipid bilayer than integral proteins. Integral proteins are tightly bound to membranes by hydrophobic forces.

Integral proteins are amphiphilic, the protein segments immersed in a membrane’s non-polar interior have predominantly hydrophobic residues, whereas those proteins that extent into the aqueous environments have polar residues.

Fro example, one of the major glycoproteins in the plasma membrane of erythrocytes is glycophorin A; a 131 amino acid protein that was the first integral protein to be sequenced. This revealed that the polypeptide chain of glycophorin consists of three domains.

1. An N-terminal region on the extracellular side of the membrane that contains all the N-and O-linked glycosylation sites;
2. A hydrophobic central region that is buried in the hydrophobic core of the bilayer; and
3. A C-terminal region rich in polar and charged residues that is exposed on the cytosolic side of the membrane.
From the above explanation,it is clear that for the proper reconstitution of the protein, length of hydrophobic segment of the protein should be increased. That is, the integral proteins should have a hydrophobic centre region at the central region of the plasma membrane. The C-terminal region rich in polar residues should be exposed on the cytosolic side of the membrane. For this, the length of hydrophobic segment should be increased.

2.The tetrapeptide “KDEL” is well known as a retrieval signal of several newly synthesized proteins. This process is mediated through specific receptor– KDEL interaction. Any single amino acid change in this tetrapeptide is not allowed in terms of its binding with its receptors and its subsequent retention in specific organelle whereas, secretory proteins are devoid of such tetrapeptide. From this observation indicate the localization of the receptor of this terapeptide.
1. Plasma membrane
2. Golgi
3. Endoplasmic reticulum
4. Mitochondria
Ans:- 2
Expln:- Retaining and Retrieving Resident ER Proteins What determines, whether a particular protein in the membrane of the ER remains in the ER or procedes on to the Golgi complex ? Studies suggest that proteins are maintained in an organelle by a combination of two mechanisms.

1. Retention of resident molecules that are excluded from transport vesicles. It may be based primarily on the physical properties of the protein. For example, soluble proteins that are part of large complexes or membrane protein with short transmembrane domains are not likely to enter a transport vesicle.

2. Retrieval of “excaped” molecules back to the compartment in which they normally reside.

Proteins that normally reside in the ER, those both in the lumen and in the membrane, contain short amino acid sequences at their C-terminus that serve as retrieved signals, ensuring their return to the ER if they should be accidentally carried forward to the Golgi complex. The retrieval of “escaped” ER proteins from these compartments is accomplished by specific receptors that capture the molecules and return them to the ER in COPI coated vesicles. Soluble resident proteins of the ER lumen posses the retrieval signal “lys-asp-glu-leu” (or KDEL in simple-letter nomenclature). These proteins are recognized and returned to the ER by the KDEL receptor, an integral membrane protein that shuttles between the cis Golgi and the ER compartments. If the KDEL sequence is deleted from and ER protein the excaped proteins are not returned to the ER but instead are carried forward through the Golgi complex. Coversely, when a cell genetically engineered to expressed a lysosomal or secretory proteins that contains an added KDEL C-terminus, that protein is returned to the ER rather than being sent on to its proper destination.

Coat Protein Complex (COP)

COPII – coated vesicles transport proteins from the rough endoplasmic reticulum to the Golgi, whereas
COPI – coated vesicles mainly transport proteins, from the cis-Golgi back to the rough ER.

3.     125I-labelled diaminofluorene (DAF) is a well known photoactivable hydrophobic probe of plasma membrane integral protein. To determine the approximate length and number of hydrophobic domain in any integral membrane protein, a controlled experiment (following standard protocol) is carried out. In order to ascertain the aforesaid aspects indicate the correct combination of experimental protocols from the following choices:
1.   Intact membrane was allowed to interact with DAF and unincorporated DAF measured.
2.   Intact membrane was allowed to interact with DAF, lysed and total protein precipitated with TCA and amount of radioactivity incorporated in the total proteins in the TCA precipitated fraction measured
3.   Intact membrane was allowed to interact with DAF, then membrane was solubilized with detergent, digested with proteases (limited proteolysis) run on SDS-PAGE followed by autoradiography.
4.   Intact membrane was allowed to interact with DAF followed by complete proteolysis, SDS-PAGE and finally autoradiography

Ans:- 3

Expln:- Peripheral membrane proteins are less tightly bound to the lipid bilayer than integral membrane proteins and can readily removed by washing the membranes with a solution of high ionic strength or high pH. Integral proteins are tightly bound to the membrane through interactions with the hydrophobic core of the bilayer. Integral proteins can be extracted from them only by using agents that disrupt the membrane structure, such as organic solvents (eg; chloroform) or detergents.

4. Cyclins are the regulatory subunits and cyclin-dependent kinases (CDKs) are the catalytic subunits. Following diagram represents the involvement of cyclins and CDKs in various stages of cell cycle.

If we knock down cyclin D in a cell by shRNA, which one of the following graphs correctly represents the level of CDK2 activity ?

Ans:- 3

Expln:- A rising level of G1-cyclins (cyclin D) bind to their corresponding Cdk (Cdk4) and signal the cell to prepare the chromosome for replication. Since DNA replication takes place in S-phase, S-phase cyclins (cyclins E and A) bind with their corresponds Cdk (Cdk2). Cyclin A-Cdk2 complex enters the nucleus and DNA replication take place and cyclin E is destroyed. Then the mitotic cyclins (B cyclins) bind with their corresponding Cdk (Cdk1) and initiates cell-division.

If we silence cyclin D by shRNA (short hairpin RNA), Cdk2 activity and the subsequent activities will be arrested.

More Questions & Answers Refer Simple Instant Notes