Spectroscopic Properties of Aminoacids• All amino acids absorb in infrared region

• Only Phe, Tyr, and Trp absorb UV

• Absorbance at 280 nm is a good diagnostic device for amino acids

• NMR spectra are characteristic of each residue in a protein, and high resolution NMR measurements can be used to elucidate three-resolution NMR measurements can be used to elucidate three-dimensional structures of proteins

Todos os aminoácidos absorvem na região dos infravermelhos

Apenas a tirosina,o triptofano e a fenilalaninaabsorvem na região dos ultravioletas

Determinação da sequência de proteínas

Em 1953 Frederick Sanger sequenciou as duas cadeias da insulina.

• Os resultados de Sanger estabeleceram que todas as moleculas de uma determinada proteína têm a mesma sequência de aminoácidos.aminoácidos.

• As proteinas podem ser sequenciadas de duas maneiras:

- sequenciação real da cadeia polipeptidica

- sequenciação do DNA do gene correspondente

Peptide ~ 2-10 amino acidsPolypeptide ~ 10-50 amino acidsProtein ~ 50- amino acids

- How many AA sequences are there for a typical protein 100 AA long?

20100

- Proteins have a amino-end (NH2) and carboxyl-end (COOH)

- In the lab, proteins can be hydrolyzed (to aa) by strongacid treatment

- Physiologic hydrolysis by peptidases and proteases

Protein ~ 50- amino acids

Amino acid sequence is encoded by DNA Amino acid sequence is encoded by DNA base sequence in a genebase sequence in a gene

Second letter

T C A G

T

TTTPhe

TCT

Ser

TATTyr

TGTCys

T

TTC TCC TAC TGC C

TTALeu

TCA TAAStop

TGA Stop A

TTG TCG TAG TGG Trp G

CTT CCT CATHis

CGT T

First le

tter

Th

ird le

tter

C

CTT

Leu

CCT

Pro

CATHis

CGT

Arg

T

CTC CCC CAC CGC C

CTA CCA CAAGln

CGA A

CTG CCG CAG CGG G

A

ATT

Ile

ACT

Thr

AATAsn

AGTSer

T

ATC ACC AAC AGC C

ATA ACA AAALys

AGAArg

A

ATG Met ACG AAG AGG G

G

GTT

Val

GCT

Ala

GATAsp

GGT

Gly

T

GTC GCC GAC GGC C

GTA GCA GAAGlu

GGA A

GTG GCG GAG GGG G

FDNB - 1-fluoro-2,4- dinitrobenzene (FDNB)

Sanger process

Edman process

The Coplanar Nature of the Peptide Bond

• The peptide group consists of 6 atoms• Peptide bonds have some double bond properties• Virtually all peptide bounds occur in this trans configuration

Six atoms must lie in a single plane:First amino acid’s alpha carbon

Carbonyl carbon

Carbonyl oxygen

Second amino acid’s amide nitrogen

Amide hydrogen

Second amino acid’s alpha carbon

The Peptide Bond Is Rigid and PlanarPeptide torsion angles

The planar peptide group - Each peptide bond has some double-bond character due to

resonance and cannot rotate. Three bonds separate sequential carbons in a polypeptide

chain. The N-C and C-C bonds can rotate, with bond angles designated phi and psi, respectively. The peptide C-N bond is not free to rotate. Other single bonds in the

backbone may also be rotationally hindered, depending on the size and charge of the R

groups. In the conformation shown, phi and psi are 180º (or -180º). As one looks out from

the carbon, the and angles increase as the carbonyl or amide nitrogens (respectively)

rotate clockwise.

The backbone of a polypeptide chain can thus be pictured as a series of rigid planes

with consecutive planes sharing a common point of rotation at C alpha

trans versus cis - peptidestrans peptide bond cis peptide bond

Common Errors - One of the most common folding errors occurs via cis-trans

isomerization of the amide bond adjacent to a proline residue. Proline is the only

amino acid in proteins that forms peptide bonds in which the trans isomer is only

slightly favored (4 to 1 versus 1000 to 1 for other residues).

Thus, during folding, there is a significant chance that the wrong proline isomer will

form first. It appears that cells have enzymes to catalyze the cistrans

isomerization necessary to speed correct folding.

Peptide boundsphi and psi = 0prohibited conformation

By convention, both phi and psi are defined as 0 when the two peptide bonds flanking that carbon are in the same plane and positioned as shown. In a protein, this conformation is prohibited by steric overlap between an -carbonyl oxygen and an -aminohydrogen atom. To illustrate the bonds between atoms, the balls representing each atom are smaller than the van der Waals.

prohibited conformation

Bond Rotation Determines Protein Folding

Unfavorable orbital overlap precludes some combinations of phi and psi

•phi = 0, psi = 180 is unfavorable

•phi = 180, psi = 0 is unfavorable

•phi = 0, psi = 0 is unfavorable

Steric Constraints on phi & psi

Ramachandran plot for L-Ala residues

The conformations of peptides are defined by the values of psi and phi. Conformations

deemed possible are those that involve little or no steric interference. The areas shaded

dark blue reflect conformations that involve no steric overlap and thus are fully allowed;

medium blue indicates conformations allowed at the extreme limits for unfavorable atomic

contacts; the lightest blue area reflects conformations that are permissible if a little flexibility is allowed in the bond angles. The plots for other L-amino acid residues with

unbranched side chains are nearly identical. The allowed ranges for branched amino acid residues such as Val, Ile, and Thr are somewhat smaller than for Ala. The Gly residue,

which is less sterically hindered, exhibits a much broader range of allowed conformations.

Three amino acids which are very different

from others!

Proline

• No free amino group

• Very rigid

• Introduces breaks in α helices and β strands

Glycine

• Lacks a side chain• Lacks a side chain

• Can be found anywhere in Ramachandran plot

• In proteins often found in flexible regions with unusual backbone conformations

Cysteine

• Disulphides

The alpha helixes formed if the values

of phi are approximately 60° and the values

of psi are in the range of 45 to 50°.

Protein Architecture — α α α α Helix

All of the hydrogen bonds point in the same direction

along the helix axis. Each peptide bond possesses a dipole moment that arises from the polarities of the NH and CO groups, and, because these groups are all

aligned along the helix axis, the helix itself has a

substantial dipole moment, with a partial positive

charge at the N-terminus and a partial negative charge

at the C-terminus

Phi -57

Psi -47

Protein Architecture — α α α α Helix

One turn of the helix represents 3.6 amino acid residues. (A single turn of the -helix involves 13 atoms from the O to the H of the H bond. For this reason, the -helix is sometimes referred to as the 3.613 helix.)

Each amino acid residue extends 1.5 Å (0.15 nm) along the helix axis. With 3.6 residues

Single turn (5.4 A)

Four NOH groups at the N-terminal end of an -helix and four COH groups at the C-

terminal end cannot participate in hydrogen bonding. The formation of H-bonds

with other nearby donor and acceptor groups is referred to as helix capping.

nm) along the helix axis. With 3.6 residues per turn, this amounts to 3.6 x 1.5 Å or 5.4 Å (0.54 nm) of travel along the helix axis per turn.

Single turn (5.4 A)

The α helix as viewed from one end, looking down the

Longitudinal axis. Note the positions of R groups,

represented by purple spheres.

Helix-Forming and Helix-Breaking Behavior of the Amino Acids

Estrutura Secundária das ProteínasFolhas pregueadas ββββ

The strands become The strands become adjacent to each other, forming beta-sheet.

The arrangement of hydrogen bonds in (a) parallel and (b) antiparallel-pleated sheets.

aa

bb

β β β β turns

Random coil

• Not really random structure, just non-repeating

– ‘Random’ coil has fixed structure within a given protein

– Commonly called ‘connecting loop region’

– Structure determined by bonding of side chains (i.e. not necessarily hydrogen bonds)

Loops

• Connect the secondary structure elements.

• Have various length and shapes.

• Located at the surface of the folded protein and therefore may have important role in biological

• Located at the surface of the folded protein and therefore may have important role in biological recognition processes.

• Proteins that are evolutionary related have the same helices & sheets but may vary in loop structures.

Ramachandran plots for a variety of structures

Relative probabilities that a given

amino acid will occur in the three

common types of secondary structure

Tertiary Structure of Ribonuclease

Insulin is one of the smallest proteins. It is only 51 residues long and consists of two polypeptide chains.

Anticorpos

(IgG)

Desnaturação e renaturação de proteínas

ureia

Results from the alteration of Secondary,Tertiary or Quaternary Structures, however

do not alter the primary structure

Protein denaturation

hidrocloreto de guanidina

do not alter the primary structure

X-ray crystallography

crystallize and immobilize single,

perfect protein

3. Analyze diffraction pattern and produce an electron density map

The crystal is

bombarded

with X-ray beams

The collision of the beams

with the electrons creates a diffraction pattern

The interaction of x-rays with electrons arranged in a crystal can

produce electron-density map, which can be interpreted to an

atomic model. Crystal is very hard to grow.

Crystallization

PDB: Protein Data Bank

http://www.rcsb.org/pdb/

Three-dimensional structures of large biological molecules, including proteins

and nucleic acids.

Contain sequence details, atomic coordinates, crystallization conditions, 3-D

structure neighborsG

- Human hemoglobin (1O1I)

- Collagen (1K6F)

- Azurin (1jzg)

- Immunoglobulin (1ZVO)

- Outer membrane protein (2F1T)

Protein Purification – a multi step process

Object: to separate a particular protein from all other proteins

and cell components

There are many proteins (over 4300 genes in E. coli)

A given protein can be 0.001-20% of total protein

Other components:

nucleic acids, carbohydrates, lipids, small molecules

Enzymes are found in different states and locations:

soluble, insoluble, membrane bound, DNA bound,

in organelles, cytoplasmic, periplasmic, nuclear

Protein Purification and Analysis

Dialysis

Separation of proteins based on physical

and chemical properties

• Solubility

• Binding interactions

• Surface-exposed hydrophobic residues

• Charged surface residues

• Isoelectric Point

• Size and shape

Column Chromatography: Principles

• Cation exchangers contain negatively charged polymer

• Anion exchangers contain positively charged polymer.

• Is affected by pH

Separação por carga

Cromatografia de troca iónica

• Also called gel filtration: The column

matrix is a cross-linked polymer with

pores of selected size.

• Larger protein migrate faster than

smaller ones because they are too

large to enter the pores

Size exclusion chromatography

Separação por tamanho

Cromatografia para filtração gel

size Molecular mass

(daltons)

10,000

Gel filtration chromatography

10,000

30,000

100,000

• Separate protein by their binding specificities. The proteins retained on the column are those that bind specifically to a ligand cross-linked to the beads. Proteins that do not binds to ligands are washed through to column

Affinity Chromatography

Separação por afinidade

Cromatografia de afinidade

Logarithmic relationship between the molecular mass of a protein and its relative electrophoretic mobility in SDS-PAGE.

SDS binds to most proteins in amounts roughly proportional to the molecular weight

of the protein, about one molecule of SDS for every two amino acid residues.

The bound SDS contributes a large net negative charge, rendering the intrinsic

charge of the protein insignificant and conferring on each protein a similar charge-

to-mass ratio.

MW A B C D E F G A/3 B/3 D/3

ββ’

225

σ32

50

35

10 kDa

Two-dimensional (2D) gel electrophoresis