Proteins Part I:  Protein Structure and Digestion

Proteins Part I:  Protein Structure and Digestion

 

  1. Laboratory Objectives:

Students will:

  • Review the central dogma of biology and understand how proteins are made from DNA/RNA templates.
  • Build a model of one of the peptide bonds present in your polypeptide chain based on your derived amino acid sequence.
  • Understand how enzymes such as protease and pepsin act to digest proteins in the body using a gelatin experiment.
  • Observe protein digestion in gelatin wells treated with different enzymes and controls and record results.

 

Laboratory report due next class period:

  • Data Record and Discussion Questions (Sections D, E, and F)

 

B. Introduction

Proteins

Proteins are a group of compounds that are made up of one or more strands of amino acids (polypeptide chain).  Amino acids are the building blocks of proteins. Each amino acid contains a carbon atom bound to a hydrogen atom (H), an amino group (NH2), an acid group (COOH), and a side chain.  Like carbohydrates and lipids, proteins contain the elements carbon, hydrogen, and oxygen, but proteins are unique in that they also contain the element nitrogen in their structure.

 

Central Dogma of Biology:  DNAà RNA à Protein

The amino acid chains of each type of protein molecule contain a characteristic number of amino acids that are bound together in a precise order.  The genetic code or DNA (deoxyribonucleic acid) specifies 20 different amino acids commonly found in proteins.  DNA is made up of nucleotide bases (ATCG) that pair up in the following way:  Adenine–Thymine and Guanine–Cytosine.  A stretch of DNA that provides the blueprint for the structure of a polypeptide chain is called a gene.  DNA contained in the nucleus of each cell must first be transcribed into RNA before protein synthesis can occur.  RNA is also made up of nucleotide base pairs except that in the place of thymine, RNA strands contain uracil (AUCG).  Messenger RNA (mRNA) sequences transcribed in the nucleus will then move into the cytoplasm where they can be translated into the specific amino acid sequence needed for that particular polypeptide chain.  Each mRNA strand is translated with the help of ribosomes.  The mRNA nucleotide sequence contains three-letter codons.  Each codon corresponds to a particular amino acid (see Table 1).  As the mRNA sequence is read by the ribosomal machinery, each amino acid is added until the entire polypeptide chain is formed.

 

Protein Structure

            Amino acids are joined end-to-end during protein synthesis by the formation of peptide bonds.  Peptide bonds are formed in a condensation reaction between the nitrogen atom in the amino group of one amino acid and the carbon atom in the acid group of the next amino acid.  A molecule of water is lost when the peptide bond is formed.  Many amino acids bonded together constitute a polypeptide.  A protein is made of one or more polypeptide chains folded into a complex 3-dimensional shape.  Folds and bends in the chain occur when some of the amino acids attract each other and other amino acids repel each other.  After polypeptide chains are synthesized and have folded, several may bind together to form the final protein, whose function may also depend on the addition of a metal ion or a vitamin molecule.

 

Protein Digestion

            Protein consumed in the diet provides the raw material to make all the various types of proteins that the body needs.  Protein must be broken down to amino acids before entering the bloodstream.  Protein digestion begins in the stomach, where hydrochloric acid denatures proteins, opening up their folded structures (3-dimensional structure) to make them more accessible to enzyme attack.  Stomach acid also activates the protein-digesting enzyme, pepsin (from the inactive form, pepsinogen) which breaks protein into polypeptides and amino acids.  When the polypeptides enter the small intestine, they are broken down into smaller peptides and amino acids by a host of pancreatic protein-digesting enzymes such as trypsin, chymotrypsin, and carboxypeptidases.

 

Gelatin Experiment

Proteins are hydrolyzed by enzymes collectively referred to as proteases, each of which is able to break the bonds in proteins in a specific way.  In today’s experiment, the protein gelatin will be used to visualize protein digestion.  Gelatin is a protein that is the gelling component of Jell-O.  Enzymes like proteases digest (or break the peptide bonds in) the protein chain so that the broken protein can no longer gel.  The Jell-O will liquefy when it is exposed to these enzymes.  Many detergents and household cleaners have added enzymes like proteases that break down or digest a variety of organic molecules.  In this lab activity, you will observe how much digestion occurs in gelatin plates when treated with various enzymes (derived from animal tissues) and various household detergents (with added enzymes).

           

  1. Methods

C1:  Lab Preparation for Protein Digestion Experiment

1.) Obtain a Petri dish with gelatin from the refrigerator (TA will provide you with a

Petri dish). Label your plate with your group number.

2.) Use a straw to carefully cut holes in the gelatin.  Pull out the straw very

carefully. Repeat this step for a total of 12 holes arranged on your plate as

follows:

 

 

 

 

 

 

  • Once all of the wells have been cut with a straw as shown above, use a toothpick to carefully remove the gelatin from each well. This should make 12 holes in your dish.
  • Label each well using your red pencil with a number 1-12 on the bottom of your plate.
  • Use a ruler to measure the diameter of each well in millimeters (mm) and record the initial diameter for each well in the table provided in Section E of your data record.
  • Load each well with its treatment by using your plastic disposable pipette to add 1 drop of the following reagents to the corresponding duplicate wells:
Well Number Treatment (4 drops)
1 & 2 Water alone
3 & 4 Protease solution
5 & 6 Trypsin solultion
7 & 8 Hydrochloric acid (1 N HCl)
9 & 10 Unknown 1
11 & 12 Unknown 2

 

  • Place the lid on your gelatin plate and carefully give it to your TA to be stored in the refrigerator for 1 hour.
  • Go to Section E, number 1 and predict how much digestion you expect to occur in each well. Make sure you illustrate which wells were negative versus positive controls.
  • While the plates are incubating, move on to Section C2 for the DNA-RNA-Protein exercise.

 

C2:  DNA transcription and translation—Building a Protein

 

  • You will be provided (TA) with a random sequence of DNA on a small sheet of paper.
  • Based on your knowledge of DNA-RNA base pairing, you must transcribe your DNA sequence (ATCG) into an mRNA sequence (AUCG). Fill in Section D, number 1 with your transcribed mRNA sequence.
  • Identify the start codon in your sequence (the codon that codes for the amino acid, Methionine is your start codon) and, then, compile a list of all of the codons in your mRNA sequence.
  • Use Table 1 to determine which amino acid corresponds to each codon in your mRNA sequence and translate your mRNA sequence into an amino acid sequence.
  • List the amino acid sequence of your polypeptide chain in the space provided in Section D.
  • Use the amino acids model set at your table and build a model of at least one of the peptide bonds contained in your polypeptide chain. Please show your TA your completed peptide bond model.
  • Answer all of the questions in your Section D data record and staple your original DNA sequence to the top of your data record in the space provided.
  • Now, move on to Section C3 to complete the Protein Digestion assay with your gelatin plate.

 

 

Table 1:  Amino acids and their corresponding mRNA 3-letter codons.

 

C3:  Protein Digestion Analysis

  • Remove your gelatin plate from the refrigerator (ask your TA for help).
  • Observe if any digestion has taken place in the wells of your plate.
  • Use the ruler to measure the diameter of each well and record your data in the table provided in Section E.
  • Calculate the change in diameter for each control well and for each experimental well. Record your results in the table in Section E.
  • Assess how much digestion (using the scale in Section E, number 2) took place in each well. Is the amount of digestion what you predicted would occur?
  • Which wells had the most protein digestion? Did your unknown treatments cause protein to be digested? If so, make sure you ask your TA the identity of your unknown solutions. Why do these reagents digest protein? What ingredients do these substances have that allow them to digest protein?
  • Record all of your data and answer the discussion questions in Section F.

Laboratory Report for Protein Synthesis and Digestion

Student  Name:_______________________ Date:______________

TA Name: __________________________    

  1. Data Record (20 points)

Please attach below your original DNA sequence provided by your TA by stapling it (folded) in the box below (If you do not provide your original DNA sequence, you will lose 20 points).

 

 

 

 

 

 

 

  1. Please provide the corresponding mRNA sequence for the DNA sequence of your protein molecule (5 points).

 

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

 

  1. Where does DNA transcription simulated above take place in the cell? _________ (1 pt)
  2. How many codons (after start codon) are there in your RNA sequence? ____ (2 pts)
  3. Use the amino acid/codon information provided in Table 1 to determine the amino acid sequence of your polypeptide/protein. Please detail in the space provided below the amino acids present in your protein. Example:  Phe – Try- Gln – Pro – etc., etc. (5 points)

 

 

 

 

 

 

 

 

 

  1. How many peptide bonds does your polypeptide chain contain? ____ (1 pt)
  2. Where does RNA translation into polypeptides take place in the cell? __________ (1 pt)
  3. Name at least three components of cell machinery, molecules, or organelles that are necessary for RNA translation/Protein synthesis to take place (2 pts):  ________________________________________________________________________________________________________________________________________________
  4. How would stop codons affect the translation process for this polypeptide? Did your sequence contain any stop codons? If so, what were they? (3 pts) ________________________________________________________________________________________________________________________________________________
  1. Digestion Data Record and Analysis (10 points)
  2. In the space provided below, please write the treatment for each control and experimental well on your gelatin plate. Predict how much digestion you expect in each well based on the treatment for that well on a scale of (-) no digestion to (+++) significant digestion (5 pts).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  1. Please complete the table below for your Protein Digestion experiment. List the well number, treatment used, initial diameter (should be the same for all wells), and final diameter (mm). Score the digestion in each well using the following scale: +++ significant digestion (increase of 2mm or more), ++ moderate digestion (increase of at least 1mm), + some digestion (increase of less than 1 mm in diameter), and – no digestion occurred. (5 pts)

 

Well Number Treatment Initial Diameter (mm) Final Diameter (mm) Change in Diameter (mm) Digestion Score
1          
2          
3          
4          
5          
6          
7          
8          
9          
10          
11          
12          
  1. Discussion Questions (20 points)

 

  1. Describe in detail the sequence of events that takes place within a cell when a polypeptide chain is synthesized (DNAà RNAà Protein). Be specific. (5 pts)  ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

 

  1. List at least three different proteases you learned about in this lab exercise. Where does each one digest protein within the gastrointestinal tract? (5 pts) ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

 

  1. Explain what happened in the gelatin wells exposed to your two unknown solutions. Did digestion take place? If so, explain why it would be beneficial to have protein-digesting additives (namely enzymes) in household cleaners and detergents. (5 pts) ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

 

  1. Summarize what the 24-hour recall earlier in the semester revealed about your personal protein intake. What high-protein foods did you consume during that 24-hour period? Did your protein intake match the DRIs? Be specific.  (5 pts)  ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

 


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