PETER PAZMANY 

SEMMELWEIS UNIVERSITYCATHOLIC UNIVERSITY 



Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework** 
Consortium leader 



PETER PAZMANY CATHOLIC UNIVERSITY 

Consortium members 

SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER 

The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund *** 
**Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben 
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INTRODUCTION TO BIOPHYSICS 

(Bevezetés a biofizikába) 

STRUCTURE OF PROTEINS 

(A fehérjék szerkezete) 

GYÖRFFY DÁNIEL, ZÁVODSZKY PÉTER 



Introduction to biophysics: Structure of proteins 

Introduction 

• 	
Proteins are linear polymers of amino acids 


• 	
Four main levels of the structure of proteinsare discerned 

– 	
Sequence of amino acids 

– 	
Local conformational elements 


– 	
Global spatial arrangement of the whole protein 


– 
Subunit structure of proteins consisting of two ormore chains 



• 	
Several secondary structural elements canform motives 

• 	
Several motives can form domains 





determine the structure and drive the foldingof proteins 
• 	
An entropic force, called hydrophobic effect isthe main contributor to the stability of proteins 

• 	
The structure of proteins can be described andstudied by means of statistical physics 

• 	
The native state of proteins is usually the statewith the lowest free energy 

• 	
The folding of proteins is often a cooperative process 

• 	
A funnel-shaped free energy landscapedescribes the folding and association ofproteins 

• 	
Dynamic properties of proteins are importantfor the function 

• 	
Amino acids are compounds containing both acarboxyl and an amino group 

• 
Amino acids forming proteins are amino acids 
in which the amino group is connected to the 









.-carbon 

• 	
Only twenty of the .-amino acids take part inbuilding up protein chains 

• 	
Protein forming amino acids can be groupedbased on their chemical properties such as hydrophobicity or acid-base properties 

• 	
Different kinds of amino acids have different side chains while their backbone atoms are the same 

• 	
A general amino acid can be seen in thefollowing figure 

• 	
The .-amino group is coloured red and the .­carboxyl group is coloured blue 

• 	
R represents the side chain 








General forms of amino acids 






Chirality of amino acids 
• 	
Amino acids -except glycine -have asymmetriccarbon atoms 

• 	
Due to asymmetric .-carbon atoms, opticalisomerism occurs, so two isomers of such amino acids exist which are mirror images of each other(this phenomenon is called chirality) 

• 	
In proteins, only the L-conformers of amino acids are found (Figure 2.) 

• 	
Amino acid derivatives forming the protein chains are often referred to as 'residues' 





L and D-amino acids 




Introduction to biophysics: Structure of proteins 

Electrical properties of amino acids 

• 	
In some pH range, amino acids can beelectrically neutral but they have dissociablegroups such as carboxyl group or amino group 

• 	
At low pH, the carboxyl group is protonatedwhile at high pH, the H+ ion dissociates from it 

• 	
At high pH, the amino group is deprotonatedwhile at small pH, it carries one more H+ ion 

• 	
There exists a pH range where both the .­carboxyl and the .-amino groups are chargedwhich is called a zwitter-ion (Figure 3.) 





Zwitter-ion 






• Several amino acids have multiple dissociable groups (see the following table) 



Dissociable groups 




Introduction to biophysics: Structure of proteins 

Groups of proteinogenic amino
acids 


• 	Amino acids can be clustered into several groups based on their chemical properties 
– 
Hydrophophobic, aliphatic 

– 
Aromatic 

– 
Uncharged polar 

– 
Positively charged 

– 
Negatively charged 






Hydrophobic, aliphatic amino acids 





Aromatic amino acids 





Uncharged polar amino acids 





Positively charged amino acids 





Negatively charged amino acids 



20 




Peptide bond 

• 	
Amino acids are connected to each other bythe peptide bond 

• 	
Peptide bond is an ester-bond between the .­carboxyl group of an amino acid and the .­amino group of another one 

• 	
The peptide bond is approximately planar dueto delocalization of electrons between singleand double covalent bonds (see the followingfigure) 

– 	
The bond between C. and amino-N by the . angle 

– 
The bond between C.and the carbon atom in the carbonyl group by the . angle 



• 	
. and . angles are called torsion angles 

• 	
The energy of a given conformation is afunction of the two torsion angles 

• 	
The plot depicting the energy versus torsionangles is called a Ramachandran plot 

• 	
On the Ramachandran plot, there are distinctareas corresponding to special local structuralelements 






The planar peptide bond 




The torsion angles 






Introduction to biophysics: Structure of proteins 

Levels of protein structure 


• 	
The primary structure of proteins is the linear sequence of amino acids forming thepolypeptide chain 

• 	
The secondary structure consists of local, oftenregular and periodic structural elements 

• 	
The tertiary structure of a protein is the globalspatial arrangement of its atoms 

• 	
Quaternary structure is the subunit structureof proteins consisting of more than one chains 




Elements of secondary structure 

• 	
Secondary structure consists of local structuralelements stabilized by interactions betweenatoms being close to each other 

• 	
The elements are determined according to thepermitted areas on the Ramachandran plotand the hydrogen bonds possible to be formed 

• 	
More such secondary structural elements canform structural motifs and supersecondaryelements 




Main types of secondary structural
elements 




• 	
Helices are produced by a translation followed by a rotation 

• 	
Several helices are found in the structures of proteins, each of which is characterized by thetorsion angles of the part of the chain forming the helix 

• 	
Most of the helices are right-handed except the.-helix of which there exists a left-handed variation 

• 	
Helices are stabilized by hydrogen bonds withinthe backbone 

• 	
Due to the electrostatic properties of aminoacids, helices have a dipole moment pointingfrom the C towards the N-terminus 






Helix types 


* The number of atoms in the ring formed by making the hydrogen bond 



.-helix 




Hydrogen bonds within .-helix 



Introduction to biophysics: Structure of proteins 
Extended structures 


• 	
Extended structures can also be considered as helices because they can be produced byapplying translations and rotations 

• 	
Extended structures are the most stretched conformations permitted by excluded volumeconstraints 

• 	
The most frequent type of extended structuresis the ß-strand 

• 	
Several ß-strands can form a ß-sheet within which the strands can be in parallel or antiparallel orientation 




Antiparallel ß-sheet 




Hydrogen bonds in antiparallel ß-sheets 




Parallel beta sheet 




Hydrogen bonds in parallel ß-sheets 




Non-periodic elements: turns and loops 

• 	
Turns are short elements usually consisting ofonly a few residues 

• 	
Turns are stabilized by a hydrogen bondformed within the turn 

• 	
Loops are longer, extended elements but theydo not have a regular structure 

• 	
Usually loops are the most flexible parts ofproteins 




Types of turns 




ß-turn 



Introduction to biophysics: Structure of proteins 


Structural motifs or 
supersecondary elements 


• 	
Secondary structural elements can assembleto bigger units called motifs or supersecondaryelements 

• 	
Only a small number of such motifs are knownbut they can be found in a huge number of structures 

• 	
Supersecondary elements together can formstructural domains 




Introduction to biophysics: Structure of proteins 
Supersecondary elements 


• 	
The tertiary structure of proteins is the globalarrangement of their secondary structureelements, stabilized by interactions betweenelements far away from each other along the sequence 

• 	
By tertiary structure, the main classes of proteins are: globular, fibrillar and membraneproteins 

• 	
These types of structures are stabilized by thesame covalent bonds and non-covalent interactions 





Structural domains 

• 	
The most important structural unit of proteinsis the domain 

• 	
Domains are defined as portions of structurewithin which there are more interactions than between them 

• 
Structural domains usually correspond to
folding units and even functional units 


• 	
Structural classifications of proteins are basedon domains 





Structural classification of proteins in the
CATH database 





Mainly . architecture types 


47 


Mainly . architecture types 








Introduction to biophysics: Structure of proteins 

Mainly ß architecture types 



Mainly ß architecture types 









Table 8. 




Introduction to biophysics: Structure of proteins 



54 


Few secondary structures 






Quaternary structure 

• 	
Several protein chains together can form together a non-covalent complex, called anoligomer 

• 	
The arrangement of such chains defines thequaternary structure of such proteins 

• 	
The sequence of chains can be identical(homooligomers) or different (heterooligomers) 




Quaternary structure of hemoglobin 




• 	
Some non-protein molecules can be attachedto the proteins 

• 	
Co-enzymes bind to enzymes via non-covalentinteractions 

• 	
Prosthetic groups bind tightly or covalently to the protein chain 

• 	
In enzymes, the cofactors are responsible forthe reaction type and the protein – theapoenzyme – is responsible for the substratespecificity 

• 	
Short-range repulsion is due to the repulsion of the orbitals of electrons 

• 	
Van der Waals interactions are attractive forces between induced dipole moments 

• 	
Short-range repulsion and van der Waalsattraction can be treated in one expression, theLennard-Jones potential 

• 	
Lennard-Jones potential contains a termcorresponding to the r-12 repulsion and a term corresponding to the r-6 van der Waals attraction 

• 
The Lennard-Jones potential is: 





Interactions stabilizing the structure 


Table 9. 







...12 ..6 .
rm rm
V LJ =.-2·
rr 
where . is the depth of the potential well, r is

the distance of the two particles and rm is the distance where the potential reaches itsminimum 



The Lennard-Jones potential 





• 	
Electrostatic interactions are formed between a positively charged – for example lysine – anda negatively charged – for example aspartate –residue 

• 	
Due to the screening effect of water, theinfluence of these interactions is restricted to short distances 

• 	
The disulfide bond is a covalent bond formed by two SH-groups of cysteine residues 

• 	
Because the formation of disulfide bonds requires oxidative conditions, onlyextracellular proteins have disulfide bonds 

• 	
Disulfide bonds have an important role in thestabilization of small proteins 








Disulfide bond 





• 
Protein stability and folding are strongly
determined by the hydrophobic effect 


• 	
The hydrophobic effect is based on an entropyincrease upon the association of hydrophobic groups 

• 	
Around hydrophobic surfaces, water moleculesadopt the ordered arrangement which has alow entropy 

• 	
Reducing hydrophobic surface allows the water molecules to be released, accompanied by anentropy increase 




Introduction to biophysics: Structure of proteins 

• The increase in solvent compensates for the
decrease in the entropy of the protein chain 

• 	
Increasing the entropy decreases the free energy 

• 	
Due to hydrophobic effect, residues withhydrophobic side chains collapse to a core ofthe structure of protein 

• 	
So water-soluble proteins have a hydrophobic core and a polar surface 

• 	
Proteins with hydrophobic side chains on theirsurface have an intrinsic propensity for aggregation 





The hydrophobic effect 




Introduction to biophysics: Structure of proteins 
Protein folding 


• 	
It is known since the famous experiment of Anfinsen that the primary structure of proteins determines the spatial structure under thegiven conditions 

• 	
The structure in which proteins can performtheir physiological function is called the native state 

• 	
The native state corresponds to the global freeenergy minimum 

• 
The process through which protein chains 
reach their native state is called folding 






1916-1995) 





• 	
Native state of proteins corresponds to theglobal free energy minimum 

• 	
Assuming only three distinct conformationalstates per residue, and time of 10-13 seconds to switch between states, it would take 1.6·1027 years for an only 100 residue long peptidechain to reach its state with minimal free energy 

• 	
In real proteins, this time would be far longer because of the practically infinite number ofconformers per residue 

• 	
Contrary to this, proteins in nature reach their native state in at most a few seconds 

• 	
Based on his calculation, Levinthal postulatedpaths by which a protein folds and assumedthat not only the final structure but also thepath to it is encoded in the primary structure 

• 	
The funnel-shaped landscape of proteins willresolve this paradox 







Introduction to biophysics: Structure of proteins 
Statistical mechanical description of 
protein structure and folding 


• 
The protein chain and the solvent surrounding
it can be considered as a subsystem which is
in thermal equilibrium with the environment 


• 	
Thus, the Boltzmann distribution is valid for the microstates of the subsystem consisting ofthe protein chain and the solvent 


• 




• 	
Although the native state contains only one or a few chain conformations and thus relativelyfew microstates correspond to it, it can be themost favourable state because of its low energy 

• 	
The protein chain-solvent system has manydegrees of freedom so we are forced to makethe model of it simpler 

• 	
Chain conformation can be described by thepositions of its atoms 

• 	
Many solvent arrangements belong to a singlechain conformation 

• 	
Potential energy functions can be constructedto determine the energy of a givenconformation so the energy is the function ofdegrees of freedom 

• 	
If we average the energy over the solventarrangements, we can consider only thedegrees of freedom of the chain itself 

• 	
Degrees of freedom can be merged to a feworder parameters 

• 	
Energy can be plotted as a function of degreesof freedom or as a function of order parameters 








. 

The shape of such surfaces resembles a funnel 
• 	
Levinthal's paradox can be resolved based onthe funnel-shaped energy landscape 

• 	
Folding can proceed through several paths butevery path has its end on the bottom of thefunnel 





2D folding funnel 





Dill KA, Chan Levinthal to pathways to funnel. Nat. Struct. Biol. 4(1):10-9. 
09/10/11. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 



• 	
Some mesostates, containing one or more
microstates can be defined and a partition 
function can be calculated for it 


• 	
The free energy of mesostates can be
calculated from the partition function by 





F =-kT ln Q 

where Q is the partition function and F is the free energy 
. 

If we plot energy as a function of degrees offreedom we obtain a free energy surface 



Free energy profile of two-state folding