Biochem Exam 2
β strands may have ____ orientations in a β sheet
parallel or antiparallel
Secondary (2˚) structure
refers to local areas of repeating main chain structure stabilized by Hydrogen bonds BETWEEN BACKBONE ATOMS largely independent amino acid identity These structure appear over and over in various situations
Primary (1˚) structure
refers to the amino acid sequence that makes up the protein
Quaternary (4˚) structure
refers to the spatial arrangement of multiple polypeptide chains to form multisubunit complexes
Tertiary (3˚) structure
refers to the spatial arrangement of the scondary structural elements in the polypeptide chain
Lysine has pKa's of 2.18, 8.95, and 10.53, for its Carboxyl group, its amine, and its R group, respectively. What is the charge of Lysine at pH 7?
1
Glycine
Gly, G, nonpolar aliphatic
Aspartic acid has pKa's of 1.88, 9.6, and 3.65 for its carboxyl, amine, and R group, respectively. What is the charge of an aspartic acid molecule at pH 5.2
-1
Nucleic acids absorb most strongly at __
260 nm
Proteins can be quantified by examining the UV absorbance at ___, which is dominated by ___
280 nm, Tyr and Trp
Serine has a M.W. of 105, Tyrosine has a M.W. of 181, and Glutamine has a M.W. of 146. What is the M.W. of the polypeptide, YQS?
396
Three ways to represent the 3D structure of the small, single-chain protein ubiquitin
Cartoon Stick model and close-up Solvent-accessible surface model
What physiological effect would you predict from a mutation that replaced with serine the cysteine in the constant part of the immunoglobulin light chain that is involved in disulfide-bond formation with the heavy chain? A. loss of the antigen binding B. antigen binding specificity C. enhancement in the antigen binding D. versatility in the antigen binding
A
Steric interactions determine peptide conformation
Certain ϕ and ψ angles result in steric clashes, where atoms are closer than their van der Waals radii. These conformations are not allowed. The backbone of an α helix results in closely packed atoms that do not sterically clash.
Start codon
AUG
Alanine
Ala, A, nonpolar aliphatic
α-Amino acid structure
Amino group attached to the carbon α to the carboxylic acid
The specific interaction between an antibody and antigen occurs by virtue of both shape and ________ complementarity.
Charge
Asparagine
Asn, N, polar
Which of the following amino acids side chains would be most soluble in water?
Aspartic Acid
Average Mass of Amino Acid vs Average Mass of Amino acid in a protein
Average Weight of Amino Acids = 137 Average Weight of Amino Acids in proteins = 110 Release of water: Water weighs 18 g/mol
Which of the following is FALSE when considering the standard genetic code? A. AUG serves as the translation start codon in most cases. B. Each of the three stop codons can also encode rare modified amino acids. C. Three separate codons encode translation stop signals. D. There are 64 possible codons to represent 20 common amino acids. E. Apart from methionine, the only other amino acid with a single codon is tryptophan.
B
Which of the following statements about insulin is INCORRECT? A. It is synthesized as a random coil single chain on membrane-bound ribosomes. B. The disulphide bonds form after the final proteolytic cleavage to yield mature insulin. C. It is stored in the pancreas in an inactive form. D. The leader sequence is cleaved off after membrane transport. E. In the active form it has two polypeptide chains joined by disulphide bonds.
B
You are working to purify Snufflease, a protein with a pI of 6.7. You have at your disposal a Cation Exchange column. What pH buffer would you put your Snufflease in to make use of the column? A 6.7 B 4.5 C 8.2
B 4.5
Which of the following amino acids would most likely be found on the surface of a protein?Check all that apply. A. Phenylalanine B. Proline C. Leucine D. Valine E. Aspartic acid
B, E
Polypeptides can be separated based on
Binding activity Size Charge
Linus Pauling: Rules for secondary structure
Bond angles and lengths should be like those for respective free amino acids No atoms should approach one another more closely than allowed by their van der Waals radii Amide group must remain planar Noncovalent bonds (hydrogen bonds) stabilize 3-D structure
SDS gel electrophoresis can be used to determine: A. the overall charge on a polypeptide. B. whether subunits in a protein complex are identical or not. C. the molecular mass of denatured protein subunits. D. the molecular mass of a native protein complex. E. none of the above.
C
Which of the following is CORRECT when considering the tertiary structure of globular proteins? A. All parts of the proteins can be classified as helix, β sheet or turns. B. β sheets cannot be twisted or wrapped into barrel structures. C. Hydrophobic residues are normally on the inside and hydrophilic residues are on the outside. D. The amino acid proline never occurs in a region where the polypeptide chain bends or turns. E. None of the above.
C
Which of the following is NOT true of immunoglobulin molecules? A. Some are membrane bound. B. They have two identical heavy chains and two identical light chains. C. Antigenic determinants reside only in the variable region of the light chains. D. Proteolytic cleavage can generate fragments containing the antigen-binding site. E. They consist of four polypeptide chains held together by disulphide bridges.
C
Cyanogen bromide (CNBr)
Cleaves proteins specifically at methionyl residues
Hydrophobic side chains
Cluster about the hydrophobic heme cofactor and on the side of the molecule
Protein folding is a thermodynamically favorable process under physiological conditions because: A. no intermediate stage disulphide bonds form during the folding process. B. there is an increase in entropy associated with protein folding. C. there is a decrease in entropy of the solvent by burying hydrophobic groups within the molecule. D. of the large negative enthalpy change associated with many noncovalent interactions. E. all of the above.
D
The protein that makes up about a third of the total protein mass in animals is: A. hemoglobin. B. myoglobin. C. β-keratin. D. collagen. E. α-keratin.
D
L-alanine is the mirror image of __
D-alanine (Note the Fischer representations are also mirror images)
Ion exchange resins
DEAE (diethylaminoethyl) cellulose and CM (carboxymethyl) cellulose are widely used. At a pH above the isoelectric point a protein carries a negative charge and will bind to DEAE-cellulose. Protein can be selectively eluted by applying a gradient of pH or increasing ionic strength.
Transcription and translation summary
DNA 5'-3': Same as mRNA DNA3'-5': Complement to mRNA (templatte)
Applications of mass spectrometry include: A. determining the primary structure of proteins. B. detection of post-translational modifications on proteins. C. determination of the mass of a protein. D. A and B. E. A, B, and C.
E
Common features of folded globular proteins
Globular proteins have a nonpolar (hydrophobic) interior and a more hydrophilic exterior β sheets are usually twisted or wrapped into barrel structures. The polypeptide chain can turn corners, e.g., β turns:
Which Amino Acids must a protein have to be detected by absorbance at 280 nm?
F, W, Y (aromatics)
At pH=0, the net charge on a polypeptide will be negative. True or False.
False
Proteins cannot self-assemble into a functional conformation after they have been denatured.True or False.
False
The interactions that stabilize multisubunit complexes are different to those that stabilize tertiary structure.True or False.
False
Tropocollagen is a double helix of two left-handed polypeptide chains. True or False.
False
When referring to the amino acid sequences of proteins, sequence homology is the same as sequence similarity. True or False.
False
Protein tertiary structure can be composed of varying degrees of α-helices and β-sheets
Fluorescent Protein β-sheets G-protein mixture Hemoglobin α-helices most proteins contain a mixture
Glutamine
Gln, Q, Polar
Histidine
His, H, + polar
________ chromatography is used to separate proteins based on their surface charge.
Ion exchange
An amide bond between the α-carboxylic acid group of one amino acid and the α-amino group on another is called a ________.
Peptide Bond
Formation of peptides
Peptides are small condensation products of amino acids They are "small" compared to proteins (Mw < 10 kDa)
Globular proteins fold into defined structures
Proteins have diverse structures, with varying amounts of helix, sheet and loop regions Larger proteins often contain two or more distinct "domains" of compact folded structure A typical protein "domain" is ~200 amino acids and will fold independently A domain frequently possesses some defined function (e.g., DNA recognition, oligomerization, cofactor binding, etc.)
A ________ plot describes which structures in a polypeptide are sterically possible and which are not based on the angles of rotation about the backbone Namide -Cα and Cα-Ccarbonyl bonds.
Ramachandran
Overview of size exclusion chromatography
Separation of proteins is based on the apparent sizes of the molecules
Serine
Ser, S, Polar
Classification of protein structure
Some proteins are predominantly helix or sheet, others possess a mixture of helix and sheet, or very little defined secondary structure
Not all parts of a globular protein structure can be categorized as helix, sheet, or turn.
Such regions are often called "random coil" or, more properly, "irregularly structured regions."
Ramachandran plot
The Ramachandran plot shows sterically allowed ϕ and ψ angles.
Rotation around bonds in a polypeptide backbone
The positions of the six atoms in each amide plane are essentially fixed; thus, free rotation is allowed about the Cα carbons. However, this rotation is restricted by steric interactions. Such rotation is described by two angles termed ϕand ψ
In size exclusion chromatography, the smallest proteins are eluted last.
True
Protein biosynthesis uses only L-amino acids.True or False
True
The amino acid side chain residues in an α helix point outwards away from the center of the helix.True or False.
True
The folded conformation of proteins can be stabilized by the binding of a metal ion or cofactor. True or False.
True
The immunoglobulin domain is a stable scaffold containing two antiparallel β- sheets upon which to display hypervariable loops.True or False.
True
Stop Codon
UAA, UGA and UAG
The pKa of a side chain is influenced by its environment
Unperturbed, the pKa of glutamic acid is 4.2 In proximity to a negative charge, the pKa of the glutamic acid is raised In proximity to a positive charge, the pKa of glutamic acid is lowered
The peptide bond links ___
amino acids
Numbering (and naming) starts from the __ and goes to the ___
amino terminus, carboxyl terminus
R - groups
are the side chains that distinguish the 20 common amino acids
ψ
between Cα and the carbonyl C
ϕ
between N and Cα)
The ___ of two amino acids forms a peptide bond and releases ___
condensation, water (This reaction is not thermodynamically favorable and is coupled to ATP hydrolysis during protein biosynthesis)
Conservative mutations
conserve the chemical property (e.g. charge) and/or the size of the amino acid
polypeptides
contain greater than 15-20 residues.
At physiological pH, the carboxylic acid group of an amino acid will be ________, while the amino group will be ________, yielding the zwitterion form.
deprotonated, protonated
Larger proteins elute ___ because they are excluded from the interior volume of the chromatography matrix
earlier
L-alanine and D-alanine are ___
enantiomers
Proteases
enzymes that cleave specific peptide bonds
All amino acids have a chiral α-carbon EXCEPT ________.
glycine
All α-amino acids, except __, contain an asymmetric α-carbon
glycine
The side chain of ________ has a pKa in the physiological pH range and is therefore often involved in proton transfer during enzymatic catalysis.
histidine
nonconservative mutations
involve larger differences in polarity/size
Proline in helices but good in beta turns because
is destabilizing, because it puts a kink in backbones
Glycine in helices but good in beta turns because
is mildly destabilizing in helices because it has no R group, therefore no constraint on angles it can adopt.
Smaller proteins elute ___ because they are retained within the pores of the chromatography matrix, resulting in a longer residence time on the column
later
As pH increases, the overall charge on a peptide will become more
negative
Side chain positions in a β sheet
neighboring side chains are located on opposite faces of the sheet, which is stabilized by main-chain hydrogen bonds between adjacent β strands.
Oligopeptides
peptide chains consisting of only a few residues
Delocalization of electrons creates a
planar and stable peptide bond
As pH decreases the overall charge on a peptide will become more
positive
When I tell you a protein is MPQRNSTSKLVALDEK, what type of structural information is that?
primary
Evolutionary relationships are inferred from ___
protein amino acid sequences
At neutral pH (~7) the amino acid nitrogen is __ and the carboxylic acid is __ to yield the __
protonated, deprotonated, zwitterion
Peptides are stable unless ___ is present
strong acid or a catalyst
Hyrophillic Side Chains
tend to lie on the solvent exposed surface of the protein
Protein tertiary structure is characterized by
the content of helix and sheet secondary structures as well as defined turns that link these secondary structures
Side chain positions in an α helix
the side chains radiate away from the helical axis. The center of the α helix consists backbone atoms, closely packed. hydrogen bonds that stabilize the helix
When R is not a hydrogen atom,
the α-carbon is an asymmetric center
Peptide bond cleavage
ΔG˚' for peptide bond hydrolysis ~ -10 kJ/mol •6N HCl at high temperature will degrade proteins to free amino acid
Cysteine Side Chains can form Disulfide Bridges
•Disulfide bridges exist in Oxidizing Environments •Like the endoplasmic reticulum and the extracellular environment •Reducing Environments disrupt disulfides •Like the cytosol