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Chapter 22: Problem 34

Ribosomal proteins can be separated by two-dimensional electrophoresis (a technique that separates proteins based on both charge and size). One of the proteins in the large ribosomal subunit is sometimes acetylated at its N-terminus. Explain why two-dimensional electrophoresis of ribosomal proteins yields two spots corresponding to this protein.

Short Answer

Expert verified
Two spots appear due to acetylation changing the protein's charge, not size.

Step by step solution

01

Understanding the Basics of Two-Dimensional Electrophoresis

Two-dimensional electrophoresis separates proteins on two independent axes: by charge (isoelectric point) and by size (molecular weight). This technique typically produces a distinct spot for each unique protein form, allowing for the analysis of post-translational modifications, like acetylation.
02

Effect of Acetylation on the Protein's Charge

Acetylation adds an acetyl group to the N-terminus of the protein. This modifies the protein's charge by neutralizing a positive charge at the N-terminus, altering its isoelectric point, and potentially shifting its position horizontally (charge axis) in the electrophoresis gel.
03

Impact of Acetylation on Protein Separation

Since the acetylated and non-acetylated forms of the protein will have different isoelectric points due to the charge change from acetylation, they will separate horizontally on the first dimension of the gel based on charge.
04

Size Determination in the Second Dimension

In the second dimension, proteins are separated based on size. The acetyl group modifies the charge but not the size significantly, so the two protein forms (acetylated and non-acetylated) should align vertically, indicating they are of the same molecular weight.
05

Conclusion on the Appearance of Two Spots

On the gel, two spots will appear for the protein: one for the non-acetylated form and one for the acetylated form. Each spot corresponds to a different isoelectric point due to the presence or absence of the acetyl group, confirming the modification.

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Key Concepts

These are the key concepts you need to understand to accurately answer the question.

Post-Translational Modifications
Post-translational modifications are chemical changes that occur to proteins after they are initially synthesized in the ribosome. One common type of modification is acetylation, where an acetyl group is added to the N-terminus of a protein. These modifications can have significant effects on a protein's function, stability, and location.
They can alter the protein's charge or conformation, influencing its interactions within the cell.
  • Acetylation is important in regulating protein activity.
  • Such modifications can affect the protein's isoelectric point (pI).
  • They can change how proteins behave in electrophoresis.
Understanding these changes is crucial in biochemical research, as they provide insights into cellular processes and regulatory mechanisms.
Protein Isoelectric Point
The isoelectric point (pI) of a protein is the pH at which the protein carries no net electrical charge. This property is critical because it affects protein solubility and interaction with other molecules. In two-dimensional electrophoresis, proteins are first separated based on their pI.
  • At pH values below the pI, the protein carries a positive charge.
  • At pH values above the pI, it carries a negative charge.
  • The protein moves through an electric field until it reaches its pI, where it stops moving.
Modifications such as acetylation can shift the isoelectric point by altering the protein's charge. In the case of the ribosomal protein from our exercise, acetylation neutralizes a positive charge leading to a shift in the pI and results in distinct separation in the gel.
Protein Separation Techniques
Protein separation techniques are essential tools in molecular biology for analyzing complex protein mixtures. Two-dimensional electrophoresis is a popular method that separates proteins based on two properties: charge and size. This technique is powerful because:
  • In the first dimension, proteins are sorted by their isoelectric point.
  • In the second dimension, they are separated by molecular weight.
  • All modifications influencing charge or size can be detected.
By resolving these properties, researchers can identify different protein forms and modifications, like those caused by acetylation. The differences in charge and size allow the precise identification of protein states, aiding in the detailed study of cellular functions.

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Most popular questions from this chapter

In 1957, Christian Anfinsen carried out a denaturation experiment in vitro with ribonuclease, a pancreatic enzyme consisting of a single 124 -amino-acid chain cross-linked by four disulfide bonds (see Problem 4.47). Urea (a denaturing agent) and 2-mercaptoethanol (a reducing agent) were added to a solution of purified ribonuclease, resulting in protein unfolding with a concomitant loss of biological activity. When urea and 2 -mercaptoethanol were removed, the ribonuclease spontaneously folded back to its native conformation and regained full enzymatic activity. Why could proper protein folding occur in this experiment in the absence of molecular chaperones?

The bacterial elongation factors EF-Tu and EF-G are essential for translation in vivo, but bacterial ribosomes can translate mRNA into protein in vitro in the absence of \(\mathrm{EF}-\mathrm{Tu}\) and \(\mathrm{EF}-\mathrm{G}\). Why are these factors not required in vitro? How does their absence affect the accuracy of translation?

Predict the effect on protein synthesis if EF-Tu were able to recognize and form a complex with fMet-tRNA Met .

A new tRNA discovered in \(E\). coli contains a uridine modified to form uridine- \(5^{\prime}\)-oxyacetic acid (cmo \({ }^{5} \mathrm{U}\) ). The modified uridine can base pair with \(\mathrm{G}, \mathrm{A}\), and U. What mRNA codons are recognized by tRNA \({ }_{c m o}^{L} \mathrm{UAG}^{2}\) ?

Multidomain proteins tend to fold better inside cagelike chaperonin structures (such as GroEL/GroES in E. coli) than with cytosolic chaperones. Explain why.
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