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Advantages of knowing amino acid sequeces
- The sequence of a protein of interest can be compared with all other known sequences to ascertain whether significant similarities existComparison of sequences of the same protein in different species yields a wealth of information about evolutionary pathwaysAmino acid sequences can be searched for the presence of internal repeatsMany proteins contain amino acid sequences that serve as signals designating their destinations or controlling their processingSequence data provide a basis for preparing antibodies specific for a protein of interestAmino acid sequences are valuable for making DNA probes that are specific for the genes encoding the corresponding proteins.
o The sequence of a protein of interest can be compared with all other known sequences to ascertain whether significant similarities exist.
CPUs do this in seconds. If the newly isolated protein is a member of an established class of protein, we can __
begin to infer info about the protein's structure and function
o Comparison of sequences of the same protein in different species yields a wealth of information about evolutionary pathways.
Sequence differences give insight into __, especially if we assume the random mutation rate of proteins over time is __-->info about __
- genealogical relationships
- when evolutionary lines diverged
o Amino acid sequences can be searched for the presence of internal repeats.
These can reveal the __. Many proteins have arisen by __ of __ follwed by __..
- history of an individual protein itself.
- duplication of primordial genes followed by their diversification
o Many proteins contain amino acid sequences that serve as signals designating their destinations or controlling their processing. A protein destined for export from a cell or for location in a membrane contains a __, a stretch of about 10 hydrophobic residues near the amino terminus that directs the protein to the appropriate membrane. Another protein may contain a stretch of amino acids that functions as a __, directing the protien to __
- signal sequence
- nuclear localization signal, directing the protein to the nucleus
o Sequence data provide a basis for preparing antibodies specific for a protein of interest.
One or more parts of the amino acid sequence of a protein will elicit an __when injected into a mouse or rabbit
o Amino acid sequences are valuable for making DNA probes that are specific for the genes encoding the corresponding proteins.
Knowing primary structure enables __. DNA sequences can be determined from __ and can then be used as __to isolate the gene encoding the protein so that the __ can be determined. The gene then can provide valuable info about the __. __ is an integral part of molecular genetics, just as __ is central to the analysis of protein structure and function
- reverse genetics
- amino acid sequences
- entire sequence of the protein
- physiological regulation of the protein
- Protein sequencing
- DNA cloning
Peptide sequences can be determined by __
The first step: __
- automated Edman degradation
- determine the amino acid composition of the peptide, which is hydrolyzed into its constituent amino acids by heating it in 6 M HCl at 110oC for 24 hours.
o The amino acids can then be separated by __, where its identity is revealed by its __ (the volume of buffer used to remove the amino acid from the column) and its quantity is revealed by __
- ion-exchange chromatography
- elution volume
- reaction with an indicator dye such as ninhydrin or fluroescamine
After conjugation to the indicator, the amino acid exhibits a color with an intensity that is proportional to its __. A comparison of the chromatographic patterns of our sample __ with that of a standard mixture of amino acids would show that the amino acid composition of the peptide is (a particular set of amino acids)
The next step is to __
Pehr Edman devised a method for __
- identify the N-terminal amino acid
- labeling the amino-terminal residue and cleaving it from the peptide without disrupting the peptide bonds between the other amino acid residues
· The Edman degradation sequentially DOES WHAT?
o __ reacts with the __ of the peptide to form a __. Then, under mildly acidic conditions, a __ of the terminal amino acid is liberated, which leaves an __ shortened by __
The cyclic compound is a __amino acid, which be identified by chromatographic methods
- removes one residue at a time from the amino end of a peptide
- Phenyl isothiocyanate
- uncharged terminal amino group
- phenylthiocaramoyl derivative
- cyclic derivative
The Edman procedure can then be repeated on the __, yielding another __, which can again be identified by chromatographic methods. Three more rounds of the Edman degradation will reveal the __
By repeated Edman degradations, the amino acid sequence of some 50 residues in a protein can be determined
- shortened peptide
- PTH-amino acid
- complete sequence of the original hexapeptide
Gas-phase sequenators can analyze __quantities of peptides and proteins with the use of __ to identify each amino acid as it is released
This high sensitivity makes it feasible to do what?
- high-pressure liquid chromatography
- analyze the sequence of a protein sample eluted from a single band of an SDS-polyacrylamide gel
Proteins can be specifically __ to facilitate analysis
In practice, the peptides cannot be much longer than about __, because not all peptides in the reaction mixture do what
at each step
The peptides obtained by __ are separated by some type of __. The sequence of each purified peptide is then determined by the __. The order, however, is not yet known but obtained from overlap peptides
A second enzyme is used to split the polypeptide chain at different linkages
- cleaved into small peptides
- 50 residues
- release the amino acid derivative
- specific chemical or enzymatic cleavage
- Edman method
Additional steps are necessary if the initial protein sample is actually __. __under reducing conditions should display the number of chains
Alternatively, the number of distinct __ could be determined. After a protein has been identified as being made up of two or more polypeptide chains, __ are used to dissociate chains held together by noncovalent bonds
The dissociated chains must be separated from one another before sequence determination can begin
- several polypeptide chains.
- SDS-gel electrophoresis
- N-terminal amino acids
- denaturing agents
Polypeptide chains linked by __ are separated by reduction with thiols such as __ or __. To prevent the cysteine residues from recombining, they are then __ with __ to form stable S-carboxymethyl derivatives. Then, sequencing can happen
- disulfide bonds
- beta-mercaptoethanol or dithiothreitol
- alkylated with iodoacetate
o Genomic and Proteomic methods are complementary
Thousands of proteins have been sequenced by the __ of peptides derived from specific cleavages. But, for sequencing proteins with more than 1000 residues, __—long stretches of DNA can be cloned and sequenced, and the nucleotide sequence can be translated to reveal the amino acid sequence of the protein encoded by the gene
- Edman degradation
- recombinant DNA technology is used
Many proteins undergo __ after their syntheses; and, so DNA base sequences reveal the nascent protein before modifications
Some ends are __, others have __; __residues are oxidized to form __, side chains are altered, etc.
- posttranslational modifications
- cleavage of a larger initial polypeptide chain
- S-S links
§ Amino acids have a lot of information, but don’t show modificationsà __
· Thus, genomic and proteomic analyses are complementary approaches for __
- chemical analyses
- elucidating the structural basis of protein function