Exam 3: Amino Acids, peptides, and Proteins

You are trying to determine the sequence of a protein that you know is pure.Give the most likely explanation for each of the following experimental observations.You may use a simple diagram for your answer. (a)The Sanger reagent (FDNB,fluorodinitrobenzene)identifies Ala and Leu as amino-terminal residues,in roughly equal amounts. (b)Your protein has an apparent M_r of 80,000,as determined by SDS-polyacrylamide gel electrophoresis.After treatment of the protein with performic acid,the same technique reveals two proteins of M_r 35,000 and 45,000. (c)Size-exclusion chromatography (gel filtration)experiments indicate the native protein has an apparent M_r of 160,000.

Titration of valine by a strong base,for example NaOH,reveals two pK's.The titration reaction occurring at pK₂ (pK₂ = 9.62)is:

A nonapeptide was determined to have the following amino acid composition: (Lys)₂, (Gly) ₂, (Phe) ₂,His,Leu,Met.The native peptide was incubated with 1-fluoro-2,4-dinitrobenzene (FDNB)and then hydrolyzed;2,4-dinitrophenylhistidine was identified by HPLC.When the native peptide was exposed to cyanogen bromide (CNBr),an octapeptide and free glycine were recovered.Incubation of the native peptide with trypsin gave a pentapeptide,a tripeptide,and free Lys.2,4-Dinitrophenyl-histidine was recovered from the pentapeptide,and 2,4-dinitrophenylphenylalanine was recovered from the tripeptide.Digestion with the enzyme pepsin produced a dipeptide,a tripeptide,and a tetrapeptide.The tetrapeptide was composed of (Lys) ₂,Phe,and Gly.The native sequence was determined to be:

Provide a brief definition for a polymorphic protein.

Draw the structure of Gly-Ala-Glu in the ionic form that predominates at pH 7.

The first step in two-dimensional gel electrophoresis generates a series of protein bands by isoelectric focusing.In a second step,a strip of this gel is turned 90 degrees,placed on another gel containing SDS,and electric current is again applied.In this second step:

Lys residues make up 10.5% of the weight of ribonuclease.The ribonuclease molecule contains 10 Lys residues.Calculate the molecular weight of ribonuclease.

Hydrolysis of peptide bonds is an exergonic reaction.Why,then,are peptide bonds quite stable?

At pH 7.0,converting a glutamic acid to $\gamma$ -carboxyglutamate,will have what effect on the overall charge of the protein containing it?

For the study of a protein in detail,an effort is usually made to first:

How can a polypeptide have only one free amino group and one free carboxyl group?

Two amino acids of the standard 20 contain sulfur atoms.They are:

How are "signature sequences" useful in analyzing groups of functionally related proteins?

Draw the structures of the amino acids phenylalanine and aspartate in the ionization state you would expect at pH 7.0.Why is aspartate very soluble in water,whereas phenylalanine is much less soluble?

Which of the following describes the overall three-dimensional folding of a polypeptide?

A biochemist wishes to determine the sequence of a protein that contains 123 amino acid residues.After breaking all of the disulfide bonds,the protein is treated with cyanogen bromide (CNBr),and it is determined that that this treatment breaks up the protein into seven conveniently sized peptides,which are separated from each other.It is your turn to take over.Outline the steps you would take to determine,unambiguously,the sequence of amino acid residues in the original protein.

Amino acids are ampholytes because they can function as either a(n):

Identify the pair of peptides below that cannot be distinguished by tandem mass spectrometry.

The peptide alanylglutamylglycylalanylleucine has: