aminoacidos pepridos proteinas

8/14/2019 aminoacidos pepridos proteinas

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CHAPTER 3

Amino Acids, Peptides,

Proteins

Structure and naming of amino acids

Structure and properties of peptides

Ionization behavior of amino acids and peptides Methods to characterize peptides and proteins

Learning goals:


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Proteins:

Main Agents of Biological Function

Catalysisenolase (in the glycolytic pathway)

DNA polymerase (in DNA replication)

Transporthemoglobin (transports O2in the blood)

lactose permease (transports lactose across the cell membrane)

Structurecollagen (connective tissue)

keratin (hair, nails, feathers, horns)

Motionmyosin (muscle tissue)

actin (muscle tissue, cell motility)


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Proteins serve a wide range of

biological functions


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Amino Acids:

Building Blocks of Protein

Proteins are linear heteropolymers of -amino acids

Amino acids have properties that are well-suited to carryout a variety of biological functions

Capacity to polymerize

Useful acid-base properties

Varied physical properties Varied chemical functionality


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Most -amino acids are chiral

The -carbon always has four substituents and istetrahedral

All (except proline) have:

an acidic carboxyl group

a basic amino group

an -hydrogen connected to the -carbon

The fourth substituent (R) is unique In glycine, the fourth substituent is also hydrogen


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Amino Acids: Atom Naming

Organic nomenclature: start from one end

Biochemical designation:

start from -carbon and go down the R-group


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All amino acids are chiral (except glycine)Proteins only contain L amino acids


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Amino Acids: Classification

Common amino acids can be placed in five basicgroups depending on their R substituents:

Nonpolar, aliphatic (7)

Aromatic (3)

Polar, uncharged (5)

Positively charged (3)

Negatively charged (2)


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These amino acid side chains absorb UV light at 270280 nm


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These amino acids side chains can form hydrogen bonds.

Cysteine can form disulfide bonds.


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Uncommon Amino Acids in Proteins

Not incorporated by ribosomes

except for Selenocysteine

Arise by post-translational modificationsof

proteins Reversible modifications, especially

phosphorylation, are important in regulation and

signaling


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Modified Amino Acids Found in Proteins


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Reversible Modifications of Amino Acids


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Ionization of Amino Acids

At acidic pH, the carboxyl group is protonated andthe amino acid is in the cationic form.

At neutral pH, the carboxyl group is deprotonatedbut the amino group is protonated. The net charge iszero; such ions are called Zwitterions.

At alkaline pH, the amino group is neutral NH2andthe amino acid is in the anionic form.


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CationZwitterion Anion


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Chemical Environment Affects pKaValues

-carboxy group is much more acidic than in carboxylic acids

-amino group is slightly less basic than in amines


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Amino acids can act as buffers

Amino acids with uncharged side chains, such as glycine,

have two pKavalues:

The pKaof the -carboxyl group is 2.34

The pKaof the -amino group is 9.6

It can act as a buffer in two pH regimes.


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Buffer

Regions


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Amino acids carry a net charge of zero

at a specific pH (the pI)

Zwitterions predominate at pH values between the pKavalues of

the amino and carboxyl groups

For amino acids without ionizable side chains, the Isoelectric Point

(equivalence point, pI) is

At this point, the net charge is zero

AA is least soluble in water

AA does not migrate in electric field

2

21 pKpK

pI


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Ionizable side chains can show up

in titration curves

Ionizable side chains can be also titrated

Titration curves are now more complex

pKavalues are discernable if two pKavalues are more

than two pH units apart

Why is the side chain pKaso much higher?


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How to Calculate the pI When the

Side Chain is Ionizable

Identify species that carries a net zero charge

Identify pKavalue that defines the acid strength of this

zwitterion: (pK2)

Identify pKavalue that defines the base strength of this

zwitterion: (pK1)

Take the average of these two pKavalues

What is the pI of histidine?


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Formation of Peptides

Peptides are small condensation products of amino acids

They are smallcompared to proteins (Mw< 10 kDa)


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Peptide ends are not the same

Numbering (and naming) starts from the amino terminus

AA1 AA2 AA3 AA4 AA5

Naming peptides:


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Naming peptides:

start at the N-terminus

Using full amino acid names

Serylglycyltyrosylalanylleucine

Using the three-letter code abbreviation

Ser-Gly-Tyr-Ala-Leu

For longer peptides (like proteins) the one-

letter code can be used

SGYAL


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Peptides: A Variety of Functions

Hormones and pheromonesinsulin (think sugar)

oxytocin (think childbirth)

sex-peptide (think fruit fly mating)

Neuropeptidessubstance P (pain mediator)

Antibioticspolymyxin B (for Gram bacteria)

bacitracin (for Gram + bacteria)

Protection, e.g., toxinsamanitin (mushrooms)

conotoxin (cone snails)

chlorotoxin (scorpions)


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Proteins are:

Polypeptides (covalently linked -amino acids) + possibly:

cofactors functional non-amino acid component

metal ions or organic molecules

coenzymes organic cofactors

NAD+ in lactate dehydrogenase

prosthetic groups

covalently attached cofactors heme in myoglobin

other modifications

P l tid i d b


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Polypeptide size and number

varies greatly in proteins


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Classes of Conjugated Proteins

What to Study about Peptides and


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What to Study about Peptides and

Proteins

What is its sequence and composition?

What is its three-dimensional structure?

How does it find its native fold?

How does it achieve its biochemical role?

How is itsfunction regulated?

How does it interacts with other macromolecules?

How is it related to other proteins?

Where is it localized within the cell?

What are its physico-chemical properties?


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Column Chromatography


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Column Chromatography

Separation by Charge


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Separation by Charge

Separation by Size


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Separation by Size

Separation by Affinity


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Separation by Affinity


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Electrophoresis for Protein Analysis

Separation in analytical scale is commonly

done by electrophoresis

Electric field pulls proteins according to their

charge

Gel matrix hinders mobility of proteins according

to their size and shape


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SDS PAGE: Molecular Weight

SDS sodium dodecyl sulfate a detergent

SDS micelles bind to and unfold all the proteins

SDS gives all proteins an uniformly negative charge

The native shape of proteins does not matter

Rate of movement will only depend on size: small

proteins will move faster


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SDS PAGE can be used to calculate the


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SDS-PAGE can be used to calculate the

molecular weight of a protein

Isoelectric focusing can be used to


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Isoelectric focusing can be used to

determine the pI of a protein

Isoelectric focusing and SDS-PAGE are


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Isoelectric focusing and SDS-PAGE are

combined in 2D electrophoresis

Spectroscopic Detection of


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Spectroscopic Detection of

Aromatic Amino Acids

The aromatic amino acids absorb light in the UV region

Proteins typically have UV absorbance maxima around

275280 nm

Tryptophan and tyrosine are the strongest

chromophores

Concentration can be determined by UV-visible

spectrophotometry using Beers law: A = cl


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Specific activity (activity/total protein)


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Specific activity (activity/total protein)

can be used to assess protein purity

Protein Sequencing


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Protein Sequencing

It is essential to further biochemical analysis that we know

the sequence of the protein we are studying Actual sequence generally determined from DNA sequence

Edman Degradation (Classical method)

Successive rounds of N-terminal modification, cleavage, andidentification

Can be used to identify protein with known sequence

Mass Spectrometry (Modern method)

MALDI MS and ESI MS can precisely identify the mass of apeptide, and thus the amino acid sequence

Can be used to determine post-translational modifications

Edman s Degradation


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Edmans Degradation

MS Procedures for Sequence IDs


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MS Procedures for Sequence IDs

Protein Sequences as Clues to


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Protein Sequences as Clues to

Evolutionary Relationships

Sequences of homologous proteins from a wide rangeof species can be aligned and analyzed for differences

Differences indicate evolutionary divergences

Analysis of multiple protein families can indicate

evolutionary relationships between organisms,

ultimately the history of life on Earth

Ch t 3 S


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Chapter 3: Summary

In this chapter, we learned about:

The many biological functions of peptides and proteins

The structures and names of amino acids found inproteins

The ionization properties of amino acids and peptides

The methods for separation and analysis of proteins

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aminoacidos pepridos proteinas