Chapter 5- Proteins: Primary Structure

Increasing the salt concentration causes selective __________ (precipitation) of proteins with different solubilities.

"salting out"

C-terminal analysis is enzymatic, with carboxypeptidases -___________ -____________

- Carboxypeptidase A cleaves any residue except Pro, Arg, and Lys -Carboxypeptidase B only works on Arg and Lys

____________: Sodium Dodecyl Sulfate Protein Gel Electrophoresis. Small samples are taken from fraction tubes, SDS is added, and the samples are boiled, to denature and coat all of the amino acids with negative charges. Analytical only- no protein is recovered

SDS PAGE

Denaturation can be achieved with: (reagents for your toolbox)

Separation of chains extremes of pH; High urea concentration, urea; high guanidine HCl concentration [GuHCl); High salt concentration [NaCl]

_______ cleavage on the C-side of ______, ____________

Trypsin; Lys, Arg (basic)

one peptide chain

a monomeric protein

____________ detects the presence of proteins, but it __________________ between different proteins.

absorbance; does not distinguish

__________ liberates the amino acids of a protein

acid hydrolysis

"C alpha) is the ______ of the amino acid

alpha carbon

Peptide formation is the creation of an __________ between the carboxyl group of the first amino acid and the amino group of the next amino acid.

amide bond

"N" is the __________ of the amino acid (from the alpha-amino group)

amide nitrogen

Due to the partial double-bond character of the peptide bond, the six atoms of the peptide-bond group define a plane- the

amide plane

The peptide _________ of a protein consists of the repeated sequence :

backbone; -N-C α- C₀-

"C0" is the ________ of the amino acid (from the alpha-carboxyl group)

carbonyl carbon

In _____, this potential variety is ________ by the ___________ (<10,000 residues) and by the _______ of the polypeptide _______ into a functional structure(>40 residues)

cells; limited; efficiency of protein synthesis; ability; to fold

a protein's ionic _________, __________, ________, and ___________ ability influence its _____________ behavior

charge; polarity; size; ligand-binding; chromatographic

____________ separates based on a protein's ionic charge, polarity, size, and ligand-binding ability

chromatography

______________ is used to separate the amino acids

chromatography

the amino acid _________ of different proteins are different

compositions

This __________________ results in loss of water. What remains after water is eliminated is "residue." This, building blocks in a polymer are often referred to as residues.

condensation reaction

Subunit interactions depend on weak forces, which can be __________ (SDS denatures protein structure for electrophoresis, but no protein is recovered).

denatured

A _______________, such as _________ or ________ is needed for that.

reducing agent; DTT; beta Me

______________ separates based on protein solubility in high ammonium acetate concentration

salting out

The peptide bond adopts the ________

trans conformation

name for 3 residues

tripeptide

____________ separates based on the overall size and shape of macromolecules and larger assemblies

ultracentrifugation

Staphylococcal protease -C-side of ___________ in phosphate buffer ________ -specific for _____ in acetate (_____) and bicarbonate buffer (_____)

-Glu, Asp (acidic); pH 7 -Glu; pH 5; pH 10

Fragmentation of the chains Enzymatic fragmentation - - - Chemical fragmentation -

-trypsin -chymotrypsin -Staphylococcal protease -Cyanogen bromide

at __________, there is strong absorbance by Tyr and Trp, but not Phe.

280 nm

Disulfide reducing agents 2-Mercaptoethanol Dithiothreitol

Beta- Me DTT

Chymotrypsin

C-side of Phe, Tyr, Trp; less so Leu (large nonpolar)

Cyanogen bromide -_________ acts only on methionine residues -CNBr is useful because proteins usually have only a few ____ residues -Be able to recognize the results: a peptide with a _________

CNBr; Met; C-terminal homoserine lactone

The reaction of cyanogen bromide (_____) with a peptide results in cleavage at _____ residues and produces peptides with C-terminal _________________________

CNBr; Met; homoserine lactone residues where Met residues once were

The amino acid sequence can be determined by ___________, a procedure for removing _____________ one at a time

Edman degradation; N-terminal residues

N-terminal analysis, use ________ (phenylisothiocyanate) -derivatives are phenylthiohydantoins (___________) -leaves a new, reactive amino terminus

Edman's reagent; PTH derivatives

What's to keep thiols from reacting to form a disulfide again? Covalent modification with an alkylating agent, usually _______ (another reagent for your toolbox) irreversibly block S, __________

IOAc; preventing S-S from reforming

_______________ can identify amino acid sequences from the mass-to-charge ration of gas-phage protein __________.

Mass spectrometry; fragments

_________ separates particles on the basis of _______________.

Mass spectrometry; mass-to-charge ratio

Note that some amino acids are partially or completely ______ by acid hydrolysis, especially _______

destroyed; Trp

name for 2 residues

dipeptide

Proteins can be sequence in two ways: 1. _________amino acid sequencing 2. sequencing the corresponding DNA of the gene (today, gene databases provide these ________ protein sequences)

direct; deduced

gel _____________ and its variations can separate proteins according to ________, ________, and _______________

electrophoresis; charge; size; isoelectric point

Proteins in a small sample can be separated by _____________, based on their size, using _________. After separation, proteins are detected by staining with _______________, for visualization of "bands," representing the proteins of different sizes.

electrophoresis; semi solid-phase gel; Coomassie Brilliant Blue

__________ procedures take advantage of a protein's unique structure and chemistry in order to ______ it from other molecules

fractionation; seperate

___________ and its variations can separate proteins according to charge, size, and isoelectric point

gel electrophoresis

multiples of two or more different chains

hetermultimer

Proteins are eluted (released) by _____________ concentration

high salt

multiples of one kind of chain

homomultimer

To prevent disulfide bridges from reforming, follow with an ___________, like iodoacetate

irreversible alkylating agent; IOAc

_________ proteins have no access to the matrix of the gel beads and flow easily around the gel beads.

large

what order are proteins eluted

largest to smallest

Small-molecule targets (________, black) are immobilized through ____________________ (tan) in a column.

ligands; covalent attachment to a solid matrix

more than one

multimeric protein

____________ proteins bind anion exchanged columns

negatively charged

12-20 residues

oligopeptide

Environmental conditions such as _________ and_______ affect a protein's stability during purification

pH; temperature

Below the isoelectric point, or _______ (the _________________________) the protein will carry a net __________ charge, above the pI, a net ______ charge.

pI; pH at which there is a net charge of zero' positive; negative

The coplanar relationship of the 6 atoms in the amide group is highlighted here by an imaginary shaded _______ lying between adjacent alpha-carbons

plane

many

polypeptide

__________ proteins bind cation exchange columns

positively charged

The _______ of a protein provides information about its functions

primary structure

At that point, a _________________

protein's mobility in the gel is inversely proportional to the molecular weight

An _______ based on a protein's chemical or binding properties may be used to _______ a protein during purification

quantify

Reconstructing the Sequence -Use two or more fragmentation agents in ____________ -Sequence all the peptides produced (use Edman Degradation) -Compare and __________________ to learn the sequence of the original polypeptide chain.

separate fragmentation experiments; align overlapping peptide sequences

To be sequenced, a protein must be ________ into individual polypeptides that can be ________ into sets of ___________ fragments

separated; cleaved; overlapping

The ________ of amino acids in a protein is __________, but composition is not.

sequence; distinctive

the overall _______ and ________ of macromolecules and larger assemblies can be assessed through ___________

size; shape; ultracentrifiguration

Separation based on ________, ________________ the protein. Sample is recovered by collecting fractions.

size; without irreversibly denaturing

________ proteins are impeded by access to the inside of the gel beads, delaying travel through the column

small

Dialysis

the sample is placed in semipermeable membrane bag, and the bag is immersed in a beaker of solution (dialysate).