Course

 Instructor  
Dr.  Noor  Hassan  
Assistant  Prof.    
 
KTH,  Royal  Ins;tute  of  Technology  
Stockholm  Sweden  
 
 
Email:  n_hassank@yahoocom  
Amino  Acids,  Pep;des  and  Proteins  
 Amino  Acids  and  Pep;des    
 
Learning  goals:  

•  To  know  the  structure  and  naming  of  all  20  protein  amino  acids  

•  To  know  the  structure  and  proper;es  of  pep;des  and  the  par;cularly  the  
structure  of  the  pep;de  bond.  

•  Ioniza;on  behavior  of  amino  acids  and  pep;des  at  different  pH’s.      

•  To  know  the  general  pKa’s  of  amino  acids:    their  carboxyls,  aminos,  the  R-­‐
group  weak  acids.    
Proteins:    Enzymes,  Binding  Proteins,  Structural  
Proteins  –  all  made  from  Amino  Acids  
 Amino  Acids:    
Building  Blocks  of  Protein  

•  Proteins  are  linear  heteropolymers  of  α-­‐amino  acids  

•  Amino  acids  have  proper;es  that  are  well-­‐suited  to  carry  

out  a  variety  of  biological  func;ons  
–  Capacity  to  polymerize  
–  Useful  acid-­‐base  proper;es  
–  Varied  physical  proper;es  
–  Varied  chemical  func;onality  
Amino  acids  share  many  features,  differing  only  
at  the  R  subs;tuent  
L  and  D  forms  
 Carbon  Numbering  System  

A
 Amino  Acids:  ClassificaBon  

Common  amino  acids  can  be  placed  in  five  basic  groups  
depending  on  their  R  subsBtuents:  
•   Nonpolar,  aliphaBc  (7)  
•   AromaBc  (3)  
•   Polar,  uncharged  (5)  
•   PosiBvely  charged  (3)  
•   NegaBvely  charged  (2)  
Key to Structure. Covalent Structures and Abbreviations of the “Standard” Amino
Acids of Proteins, Their Occurrence, and the pK Values of Their Ionizable Groups
 
ConBnued  
Spectrophotometry  
UV  light  AbsorpBon  by  Proteins  –  due  to  2  Amino  Acids  
Cysteine  can  form  Disulfide  Bonds  
Uncommon  Amino  Acids  
 Amino  acids  in  Proteins  Can  be  Reversibly  Modified  

A
Non  Protein  Amino  Acids  
 Toxic  Amino  Acids  

A search for compounds producing Yunnan

Sudden Unexplained Deaths found related to
eating a mushroom.

Halford, B. C+E News Feb 13, 2012

Trogia venenata Zhu L
Which  Form  Occurs  in  Water  ?  
Glycine  Acid/Base  TitraBon  
Compare  Amino  Acids  to  Simple  Carboxylic  Acids  and  Amines  
Glutamate  has  3  pKa’s  
HisBdine  has  3  pKa’s  
 How to Calculate the pI When the Side Chain is Ionizable

•  Iden;fy  species  that  carries  a  net  zero  charge  

 
•  Iden;fy  pKa  value  that  defines  the  acid  strength  of  this  zwiUerion:  (pK2)  
•  Iden;fy  pKa  value  that  defines  the  base  strength  of  this  zwiUerion:  (pK1)    
•  Take  the  average  of  these  two  pKa  values  
 Isoelectronic  point,  pI  
 

The   isoelectronic   point   or   isoionic   point   is   the   pH   at   which   the   amino   acid   does   not  
migrate   in   an   electric   field,   i.e.   amino   acid   is   electrically   neutral,     the   zwiUerion   form   is  
dominant  (net  zero  charge)  
 
 pI  is  given  by  the  average  of  the  pKas  that  involve  the  zwi]erion.  
 Amino  acids  with  neutral  side  chains  

These   amino   acids   are   characterised   by   two   pKas   :   pKa1   and   pKa2   for   the   carboxylic   acid  
and  the  amine  respec;vely.    
The  isoelectronic  point  will  be  halfway  between,  or  the  average  of,  these  two  pKas,  i.e.      pI  
=   1/2   (pKa1   +   pKa2).   At   very   acidic   pH   (below   pKa1)   the   amino   acid   will   have   an   overall   +ve  
charge  and  at  very  basic  pH  (above  pKa2   )  the  amino  acid  will  have  an  overall  -­‐ve  charge.    
For  the  simplest  amino  acid,  
 glycine,  pKa1=  2.34  and  pKa2  =  9.6,  pI  =  5.97.  
 Amino  acids  with  acidic  side  chains  
•  The  pI  will  be  at  a  lower  pH  because  the  acidic  side  chain  introduces  an  
"extra"  nega;ve  charge.  So  the  neutral  form  exists  under  more  acidic  
condi;ons  when  the  extra  -­‐ve  has  been  neutralised.      

•  For  example:  
•  aspar;c  acid  shown  below,  the  neutral  form  is  dominant  between  pH  1.88  
and  3.65,  pI  is  halfway  between  these  two  values,  i.e.      pI  =  1/2  (pKa1  +  
pKa3),    so  pI  =  2.77.  
 Amino  acids  with  basic  side  chains  

HISTIDINE  which  has  an  extra  basic  group.  

It   has   three   acidic   groups   of   pKa's   1.82   (carboxylic   acid),   6.04  
(pyrrole  NH)  and  9.17  (ammonium  NH).    
 
His;dine   can   exist   in   the   four   forms   shown,   depending   on   the  
solu;on  pH,  from  acidic  pH  (A)  to  basic  pH.  (D).  
Star;ng  from  the  (A),  imagine  that  we  add  base,  the  most  acidic  
proton  is  removed  first  (COOH),  then  the  pyrrole  NH  then  finally  
the  amino  NH.  These  takes  us  through  each  of  the  forms  in  turn.  
At  pH  <  1.82,  A  is  the  dominant  form.  
In  the  range  1.82  <  pH  <  6.02  B  is  the  dominant  form.  
In  the  range  6.02  <  pH  <  9.17  C  is  the  dominant  form,  and  
when  pH  >  9.17,  D  is  the  major  form  in  solu;on.    
 Amino  acids  with  basic  side  chains  

 
The   pI   will   be   at   a   higher   pH   because   the   basic   side   chain   introduces   an  
"extra"  posi;ve  charge.    
 
So,  
 
his;dine,  the  neutral  form  is  dominant  between  pH  6.00  and  9.17,  pI  is  
halfway  between  these  two  values,  i.e.    pI  =  1/2  (pKa2  +  pKa3),    so  pI  =  7.59.  
 pKax    +    pKay  
pI  =   2  

+ CH3 CH3
+ CH3
H3N CO2H H3N CO2 H2N CO2 pKa1    +    pKa2  
pI  =  
H pKa1 H pKa2 H 2  
low pH (2.3) (9.7)
high pH
pI  =  6.0  

CO2H CO2H CO2 CO2

CH2 CH2 CH2 CH2 pKa1    +    pKa3  
H3N CO2H pKa1 H3N CO2 H3N CO2 H2N CO2 pI  =  
H (1.9) H
pKa3
H
pKa2 2  
(3.6) (9.6) H
low pH high pH pI  =  2.7  

NH3 NH3 NH3 NH2

(CH2)4 (CH2)4 (CH2)4 (CH2)4 pKa2    +    pKa3  
pI  =  
H3N CO2H pKa1 H3N CO2
pKa2
H2N CO2 pKa3 H2N CO2 2  
H (2.2) H H (10.5) H
(9.0)
low pH high pH pI  =  9.7  

37
Electrophoresis:    separa;on  of  polar  compounds  based  on  their  mobility  through  a  solid  
support.    The  separa;on  is  based  on    charge  (pI)  or  molecular  mass.      

+ _

+ _
_ _ _ _ + + + +
Amino  acids  are  linked  together  by  covalent  
pep;de  bonds  
 PepBde  Bond  FormaBon  

Where  does  this  occur?                                                                                                                                  Where  does  this  occur?                                                        

By   conven;on,   pep;de  
sequences   are   wriUen  
len  to  right  from  the    
N-­‐terminus  to  the  C-­‐
O R2 O R4 O R6 O terminus  
H H H H
N N N N
N N N N
H H H H
R1 O R3 O R5 O R7
 CharacterisBcs  of  pepBde  bond  
The  amide  (pep;de)  bond  has  C=N  double  bond  character  due  to  resonance  resul;ng  in  a  
planar  geometry          

O R2 _
H H O R2
N N H H restricts rotations resistant to
N + N
N N hydrolysis
R1 H O R1 H O
amide bond

The  N-­‐H  bond  of  one  amide  linkage  can  form  a  hydrogen  bond  
with  the  C=O  of  another.      
O
H N
R
N H O N H N-­‐O  distance  2.85  -­‐  3.20  Å  
O
R
N H O
R
   
H N H N op;mal  N-­‐H-­‐O  angle  is  180  °  
O O

Disulfide  bonds:    the  thiol  groups  of  cysteine  can  be  oxidized  to    
form  disulfides  (Cys-­‐S-­‐S-­‐Cys)  
1/2 O2 H2O NH2
NH2 S CO2H
2 HO2C S
SH
HO2C NH2
H2
 R6 O R8 O R10 R9 O R11 O R13
H H H H H H
N N N N N N
N N N N N N
H H H H H H
O O R9 O O O R12 O
HS S
1/2 O2

SH S
R1 O O R5 H2 R1 O O R5
H H H H H H
N N N N N N
N N N N N N
H H H H H H
O R2 O R4 O O R2 O R4 O
Structure  of  a  Simple  PepBde  
Proper;es  of  pep;de/protein  depends  on  the  amino  acid  sequence    

Gly-­‐Lys-­‐Ala  
Ala-­‐Gly-­‐Lys  

Talk  liUle,  Do  much   Do  liUle,  Talk  much  

Ser-Gly-Tyr-Ala-Leu or SGYAL
 Naming  pepBdes:    
start  at  the  N-­‐terminus  

•  Using  full  amino  acid  names  

–  Serylglycyltyrosylalanylleucine  
•  Using  the  three-­‐le]er  code  abbreviaBon  
–  Ser-­‐Gly-­‐Tyr-­‐Ala-­‐Leu  
•  For  longer  pepBdes  (like  proteins)  the  one-­‐  
le]er  code  can  be  used  
–  SGYAL  
AEGK  
 Aspartame  

A
 PepBdes:  A  Variety  of  FuncBons  
•   Hormones  and  pheromones  
–   insulin  (think  sugar)  
–   oxytocin  (think  childbirth)  
–   sex-­‐pepBde  (think  fruit  fly  maBng)  

•   NeuropepBdes  
–   substance  P  (pain  mediator)  

•   AnBbioBcs  
–   polymyxin  B  (for  Gram  –  bacteria)  
–   bacitracin  (for  Gram  +  bacteria)  

•   ProtecBon,  e.g.,  toxins  

–   amaniBn  (mushrooms)  
–   conotoxin  (cone  snails)  
–   chlorotoxin  (scorpions)  
 IntroducBon  to  PepBde  and  Protein  Structure  

primary  (1°)  structure:  the  amino  acid  sequence  

secondary  (2°):  frequently  occurring  substructures  or  folds  
ter2ary  (3°):  three-­‐dimensional  arrangement  of  all  atoms  in  a  
 single  polypep;de  chain  
quaternary  (4°):  overall  organiza;on  of  non-­‐covalently  linked    
 subunits  of  a  func;onal  protein.  

48
 Secondary  structure  of  proteins  -­‐  α  helix  

H  bond  between  the  N-­‐H  of  every  pep;de  bond  to  the  C=O  of  the  next  pep;de  
bond  of  the  same  chain.  R  groups  are  not  involved.    

(Pitch)
 Secondary  structure  of  proteins  -­‐  α  helix  
α-helix: 3.6 amino acids per coil, 5.4 Å

C   3.6  AA  
   

5.4  Å  
 
 
N  

50
Secondary  Structural  level  of  Pep;des  and  Proteins.  
β-­‐sheet:  Two  or  more  extended  pep;de  chain,  in  which  the  amide  
backbones  are  associated  by  hydrogen  bonded  

N  →  C  
an9-­‐parallel   loop   R H O R H O R H O R
or   N N N O
turn   N N N N
N  →  C   H O R H O R H O R H O

O H R O H O H R O H

N N N N
O
C  ←  N  
N N N

R O H R O H R O H R

C  ←  N  
parallel  
N  →  C  
O R H O R H O R H O

N  →  C   H
N N N N
N N N O

R H O R H O R H O R
crossover  
O R H O R H O R H O
H
N N N N
N N N O

N  →  C  
R H O R H O R H O R

N  →  C   51
 Secondary  structure  of  proteins  –  β sheet    
 
Polypep;de   chains   are   held   together   by   H   bonds   between   N-­‐H   group   of   one  
polypep;de   chain   and   C=O   group   of   the   other   chain   (e.g.   in   the   protein   fibroin   -­‐  
abundant  in  silk)    
 TerBary  Structure  level  of  Proteins  

myoglobin    
pdb code: 1WLA

Bacteriorhodopsin  
pdb  code:  1AP9  

An;-­‐parallel    
Parallel  β-­‐sheets     β-­‐sheets    
carbonic  anhydrase   of  lec;n  
pdb  code:  1QRM  
pdb code: 2LAL
 TerBary  Structure  of  polypepBdes  and  Proteins.  

Fibrous.  Polypep;des  strands  that  “bundle”  to  form  elongated    

fibrous  assemblies;  insoluble.  eg.  collagen  
 
Globular.    Proteins  that  fold  into  a  “spherical”  conforma;on.  eg  enzymes  
 
Hydrophobic  effect.  Proteins  will  fold  so  that  hydrophobic  amino    acids  are  on  the  inside  
(shielded  from  water)  and  hydrophilic  amino  acids  are  on  the  outside  (exposed  to  water)  
 

Pro • Ile • Lys • Tyr • Leu • Glu • Phe • Ile • Ser • Asp • Ala • Ile • Ile • His •Val • His • Ser • Lys
54
Primary  structure  (sequence  of  amino  acids)  determines  the  
structure  of  a  protein  
Fig.4-­‐5:  In  water,  hydrophobic  aa  cluster  inside  a  folded  
protein,  away  from  solvent.    Why?  
α helices  can  wrap  around  one  another  by  interac;ons  between  their  
hydrophobic  side  chains  to  form  a  stable  coiled-­‐coil.  [Fig.  4-­‐16]    
e.g.  α  kera;n  in  the  skin  and  myosin  in  muscles  
 Ter;ary  structure  of  proteins  
•  3D  conforma;on  or  shape  
•  Depends  on  the  proper;es  of  the  R  groups  of  
amino  acid  residues    
•  Fold  spontaneously  or  with  the  help  of  
molecular  chaperones  
•  Stabilized  by  covalent  and  non-­‐covalent  bonds  
Molecular  chaperone  proteins  assist  folding  of  other  
proteins  
[Horton  et  al.  Principles  of  Biochemistry,  2nd  ed.]  
Many  proteins  are  composed  of  separate  func;onal  domains  
e.g.  bacterial  catabolite  protein  (CAP).  Protein  domain:  a  segment  (100  –  250  aa)  of  a  
polypep;de  chain  that  fold  independently  into  a  stable  structure  [Fig.  4-­‐19]  
Noncovalent  bonds  help  protein  folding  
Covalent  disulfide  bonds  between  adjacent  cysteine  side  
chains  help  stabilize  a  favored  protein  conforma;on  
 Quaternary  structure  of  proteins  

 hemoglobin,  a  protein  in  red  blood  cells,  has  four  sub  units  (two  copies  each  of  α-­‐  and  
β-­‐globins  containing  a  heme  molecule  
 Proteins  are:    

•  PolypepBdes  (covalently  linked  α-­‐amino  acids)  +  possibly:    

•   cofactors    
-  funcBonal  non-­‐amino  acid  component  
-  metal  ions  or  organic  molecules  

•   coenzymes  
-  organic  cofactors  
-   NAD+  in  lactate  dehydrogenase  

•   prostheBc  groups  
-  covalently  a]ached  cofactors    
-  heme  in  myoglobin  
•   other  modificaBons  
 Things  to  Know  by  Now  
1.  Know  Structure  and  chemistry  of  all  20  amino  acids.  

2.  Approximate  pKa  of  amino  acid  ionizable  groups  and  their  
ionizaBon  state  at  different  pH’s.  

3.  ModificaBons  of  amino  acids  in  proteins.  

4.  Disulfide  bonds,  make  and  break  them,  and  diagram  

them.  
 
5.  Structural  levels  of  protein.