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G: PREDICTING SECONDARY AND TERTIARY STRUCTURE FROM THE PRIMARY SEQUENCE

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CHAPTER 1: LIPIDS
CHAPTER 2: PROTEINS
CHAPTER 3: CARBOHYDRATES
CHAPTER 4 - NUCLEIC ACIDS
CHAPTER 5: BINDING
CHAPTER 6 - TRANSPORT AND KINETICS
CHAPTER 7 - CATALYSIS
CHAPTER 8: OXIDATIVE-PHOSPHORYLATION
CHAPTER 9 - SIGNAL TRANSDUCTION
Chapter 10: METABOLIC PATWAYS
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How close are we to predicting the structure of a protein from it's primary structure? Not very far! However, prediction of secondary structure and regions of tertiary structure are quite good. These methods are mostly empirical and are based on the actual structures of the 10,000 or so proteins whose structures are known. This large data base has been examined to help determine rules which can be used to predict the disposition of a particular amino acid in a protein.

Secondary Structure

As we have seen previously, amino acids vary in their propensity to be found in alpha helices, beta strands, or reverse turns (beta bends, beta turns).    These difference can be rationalized from the structure of each amino acid, as described before. 

Figure:  Amino Acid Structure and propensity for secondary structure

From the data bases, propensities can be calculated to determine the likelihood that a given amino acid will be in one of those structures. Glycine for example would have a high propensity to be in reverse turns, while Pro, a helix breaker,  would have a low propensity to be in an alpha helix.   A number is assigned to each amino acid for each category of secondary structure. High numbers favor the likelihood that that amino acid would be in that structure.  The most popular list of propensities is from Chou-Fasman, where H indicates high propensity for secondary structure, h intermediate propensity, i is inhibitory, b is a intermediate breaker, and  B is a significant breaker of secondary structure.  

Chou-Fasman Amino Acid Propensities

A.A. Helix Sheet
Designation P Designation P
Ala H 1.42 i 0.83
Cys i 0.70 h 1.19
Asp I 1.01 B 0.54
Glu H 1.51 B 0.37
Phe h 1.13 h 1.38
Gly B 0.57 b 0.75
His I 1.00 h 0.87
Ile h 1.08 H 1.60
Lys h 1.16 b 0.74
Leu H 1.21 h 1.30
Met H 1.45 h 1.05
Asn b 0.67 b 0.89
Pro B 0.57 B 0.55
Gln h 1.11 h 1.10
Arg i 0.98 i 0.93
Ser i 0.77 b 0.75
Thr i 0.83 h 1.19
Val h 1.06 H 1.70
Trp h 1.08 h 1.37
Tyr b 0.69 H 1.47

 

Next a stretch of amino acids about 7 amino acids is taken, starting from the N-terminal of the protein. First the average alpha helical propensities for amino acids 1-7 are determined and assigned, let's say, to the middle (4th) amino acid in that sequence. Then alpha helical propensities for amino acids 2-8 are averaged and assigned to the middle (5) amino acid in that range. This continues until all but the first and last few amino acids have an average value assigned to them. If a contiguous stretch of amino acids has high average propensity, they are probably in an alpha helix in the native protein. This process is repeated using beta strand and reverse turn propensities. The final assignments of most probably secondary structure are made. Of course this system was tested against proteins whose tertiary structure was known. See the results for secondary structure prediction for one protein. In this example, the average propensity for four contiguous amino acids is calculated (starting with amino acids 1-4, then amino acids 5-8, etc, and continuing to the end of the polypeptide).  Next this process is repeated for contiguous stretches 2-5, 6-9, etc, and continuing to the end.

Amphiphilic Helices

Additional information about putative helices can be obtained by determining if they are amphiphilic (one side of the helix containing mostly hydrophobic side chains, with the opposite side containing polar or charged side chains.  A helical wheel projection can be made. In this a circle is draw representing a downward cross-sectional view of the helix axis.

Figure:  Helical wheel projection

The side chains are placed on the outside of the circle, staggered in a fashion determined by the fact that there are 3.6 amino acids per turn of the helix. If one side of the wheel contains predominantly nonpolar side chains while the other side has polar side chains, the helix is amphiphilic. Imagine how such helices might be packed in a protein.

Hydrophobic Structure

In a completely analogous fashion, a hydrophobic propensity or hydopathy can be calculated. In this system, empirical measures of the hydrophobic nature of the side chains are used to assign a number to a given amino acid. Many hydropathy scales are used. Several are based on the Dmo transfer of the side chains from water to a nonpolar solvent. Two commonly used scales are the Kyte-Doolittle Hydropathy and Hopp-Woods scales (used more like a hydrophilicity index to predict surface or water accessible structures that might be recognized by the immune system)

  Hydrophobicity Indices for Amino Acids

Amino Acid

 Kyte-Doolittle

 Hopp-Woods

Alanine

 1.8

 -0.5

Arginine

 -4.5

 3.0

Asparagine

 -3.5

 0.2

Aspartic acid

 -3.5

 3.0

Cysteine

 2.5

 -1.0

Glutamine

 -3.5

 0.2

Glutamic acid

 -3.5

 3.0

Glycine

 -0.4

 0.0

Histidine

 -3.2

 -0.5

Isoleucine

 4.5

 -1.8

Leucine

 3.8

 -1.8

Lysine

 -3.9

 3.0

Methionine

 1.9

 -1.3

Phenylalanine

 2.8

 -2.5

Proline

 -1.6

 0.0

Serine

 -0.8

 0.3

Threonine

 -0.7

 -0.4

Tryptophan

 -0.9

 -3.4

Tyrosine

 -1.3

 -2.3

Valine

 4.2

 -1.5

For a water-soluble protein, a  continuous stretch of amino acids found to have a high average hydropathy is probably buried in the interior of the protein.  Consider the example of bovine a-chymotrypsinogen, a 245 amino acid protein, whose sequence is shown below in single letter code.

1 CGVPAIQPVLSGLSRIVNGEEAVPGSWPWQVSLQDKTGFHFCGGSLINENWVVTAAHCGV
61 TTSDVVVAGEFDQGSSSEKIQKLKIAKVFKNSKYNSLTINNDITLLKLSTAASFSQTVSA
121 VCLPSASDDFAAGTTCVTTGWGLTRYTNANTPDRLQQASLPLLSNTNCKKYWGTKIKDAM
181 ICAGASGVSSCMGDSGGPLVCKKNGAWTLVGIVSWGSSTCSTSTPGVYARVTALVNWVQQ
241 TLAAN

A hydrophathy plot for chymotrypsinogen (sum of hydropathies of seven consecutive residues) shows many stretches that are presumably buried in the interior of the protein.

Figure:  hydrophathy plot for chymotrypsinogen

Membrane Proteins

So far we have discussed predominantly globular proteins that are soluble in water.  Proteins are also found associated with membranes.   Two major classes of membrane proteins are found in nature.

  • peripheral membrane proteins: water soluble proteins bound reversibly and non-covalently to the membrane through electrostatic attractions between charged polar head groups of the phospholipids and the protein.  These proteins can often be released from the membrane by addition of high salt, since they are often attracted to the bilayer by electrostatic interactions between charged phospholipid head groups and polar/charged groups on the protein surface.
  • integral membrane proteins: actually insert into the bilayer.  These can be released from the membrane and effectively solubilized by the addition of single chain amphiphiles (detergents) which form a mixed micelle with the integral membrane protein.  Nonionic detergents (Trition X-100, octylglucoside, etc) are often used in the purification of membrane proteins.  Ionic detergents (like SDS) not only solubilize the integral membrane proteins, but also denature them.

Figure:  Types of membrane proteins

In some of these integral membrane proteins, large extracellullar and intracellular domains of the protein are present, connected by the intramembrane regions. The intramembrane spanning region often consists of either a single alpha helix, or 7 different helical regions which zig-zag through the membrane. These  transmembrane sequences can readily be determined through hydropathy calculations.  For example, consider the integral membrane bovine protein rhodopsin.  Its 348 amino acid sequence  (in single letter code) is shown below:

MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSMLAAYMFLLIMLGFPINFLTLY
VTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLG
GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLVGWSRYIP
EGMQCSCGIDYYTPHEETNNESFVIYMFVVHFIIPLIVIFFCYGQLVFTVKEAAAQQQES
ATTQKAEKEVTRMVIIMVIAFLICWLPYAGVAFYIFTHQGSDFGPIFMTIPAFFAKTSAV
YNPVIYIMMNKQFRNCMVTTLCCGKNPLGDDEASTTVSKTETSQVAPA

 Rhodopsin hydropathy plot calculations shows that is contains seven transmembrane helices which wind through the membrane in a serpentine fashion..  

Figure:   Rhodopsin hydropathy plot


Figure:  seven transmembrane helices


Rhodopsin Hydropathy Results

No. N terminal transmembrane region C terminal type length
1 40 LAAYMFLLIMLGFPINFLTLYVT 62 PRIMARY 23
2 71 PLNYILLNLAVADLFMVFGGFTT 93 SECONDARY 23
3 113 EGFFATLGGEIALWSLVVLAIER 135 SECONDARY 23
4 156 GVAFTWVMALACAAPPLVGWSRY 178 SECONDARY 23
5 207 MFVVHFIIPLIVIFFCYGQLVFT 229 PRIMARY 23
6 261 FLICWLPYAGVAFYIFTHQGSDF 283 PRIMARY 23
7 300 VYNPVIYIMMNKQFRNCMVTTLC 322 SECONDARY 23

 

 Membrane proteins call be solubilized by addition of single chain amphiphiles (detergents).  The nonpolar tails of the detergents interact with the hydrophobic transmembrane domain of the membrane protein forming a "mixed" micelle-like structure.  Nonionic detergents like Triton X-100 and octyl-glucoside are often used to solubilize membrane proteins in their near native state.  In contrast, ionic detergents like sodium dedecyl sulfate (with a negatively charged head group) denature proteins during the solubilization process.  To study membrane proteins in a more native-like environment, proteins solubilized by nonionic detergent can be reconstituted into bilayer liposome structures using methods similar to those from Lab 1 in which you prepared dye-capsulated large unilamellar vesicles (LUVs).  However, it can be difficult to study the intra- and extracellular domains of membrane proteins in liposomes, given that one of those domains is hidden inside the liposome.  A novel technique that removes this barrier was recently developed by Sligar.  He created an amphiphilic protein disc with an opening in the center.  The inner opening is lined with nonpolar residues, while the outer surface of the disc is polar.  When the discs were added to phosphlipids, small bilayers formed inside the disc.  Membrane proteins like the b-2 adrenergic receptor could be reconstituted in the nanodisc bilayers, allowing solvent exposure of both the intracellular and extracellular domains of the receptor protein.

Figure:  Nanodisc with membrane protein

Protein Tertiary Structure

We are getting closer to predicting the tertiary structure of a protein, but as we have seen from molecular mechanics and dynamics calculations, it is a huge computational task.  There are two basic approaches which are often combined.

  • calculations using energy minimization and statistical mechanics:  These "semi-empirical" techniques don't assume any given secondary structure propensities or hydrophobicities.  Such methods have produced limited success with small proteins whose actual structure is known.
  • homology modeling based on proteins of known structure:   The structures of about 10,000  different proteins are known. This can serve as an empirical data base of possible conformations.  Instead of an infinite number of prototypical structures, it is becoming clear that there may be a reasonably low number (in the hundreds) of basic structural motifs that are used over and over in nature.  By aligning  the amino acid sequences  of different proteins, and comparing their properties (such as secondary structure propensities, hydrophobicities, etc.), probable low energy structures of the new protein can be determined.  This initial structure can be run through multiple minimization and dynamic simulations to produce a tentative "lowest" energy structure.  The structure should be compact (checked through calculation of packing density) and experimental techniques (such as spectroscopic methods) should be employed to validate the structure. 

Many models of the actual folding process have been presented, most of which have some experimental support.  In one, a hydrophobic collapse of the protein produces a seed structure upon which secondary structure and final tertiary collapse results.  Alternatively, initial formation of an alpha helix might serve as the seed structure.  A combination of the two is likely.  In one scenario, two small amphiphilic helices might form which interact through their nonpolar faces to produce the initial seed structure.

Many studies have been done on a domain of the protein villin.  A company at Stanford University (Folding at Home) actually allows you to process protein folding data on your own computer when you're not using it (an example of distributed computing).  The example below shows one simulation of length greater than 1 ms.  In the simulation, it collapses to a near native-like state then unfolds again as it iteratively probes conformational space as it "seeks" the global energy minimum.

Zhou and Karplus recently simulated the folding of residues 10-55 of Staphylococcus aureus protein A which form a 3-helix bundle structure.

Figure:   3-helix bundle

Using molecular dynamics, they carried out 100 folding simulations. Two types of folding trajectories were noted.

  • In the first type, helices form early (70% within 10 ns), but the fraction of native interhelical contacts (indicating proper packing of the helices together) and the overall packing density are not similar to the native state.  Then the helices diffuse and collide with each (in the rate-limiting step) until the native state is reached at about 19 ms.  In this model, non-obligatory intermediates can occur (due to collapse to non-native interhelical packing in the rate-limiting step) which could slow down folding.   

Figure:  helices form early


  • In another type, there is a simultaneous and quick partial helix formation and collapse (90% at 200 ns), to a state which is similar to the molten globule.  At this point, only about 20% of the native contacts are present.  The final tertiary structure is achieved after a slow process of forming native contacts within the compact state, which takes about 500 ms. 

Figure:  simultaneous and quick partial helix formation and collapse

The Fersht lab has been combining experimental and theoretical approaches to the folding/unfolding of another three helix bundle protein, Engrailed homeodomain. 

Figure:  Engrailed homeodomain

This protein is among the fastest folding and unfolding proteins known (ms time scale).  This time frame is also amenable to study through molecular dynamics simulations.  Both sets of data support a folding pathway in which the unfolded state (U) collapses in a microsecond to an intermediate state (I) characterized by significant native secondary structure and mobile side chains that is less compact than the native state (N).  The I state hence resembles the molten globule state.  To more clearly understand the unfolded state, they generated a mutant (Leu16Ala) which was only marginally stable at room temperature (2.5 kcal/mol).  Spectroscopic measurements (CD, NMR) showed this state to resemble the intermediate (I) state, with much native secondary structure and a 33% greater radius of gyration than the N state.  In effect they could study the transient intermediate of the wild type protein more easily by making that state more stable through mutagenesis.  These studies showed that the intermediate is on the folding pathway and not inhibitory to the process.  Using molecular dynamic simulations, the intermediate to native state transition was shown to proceed via a transition state (TS) in which the native secondary structure is almost all present and the helices are engaged in the final packing process.

Figure:  Complete Folding Pathway of Engrailed Homeodomain by Experiment and Simulation

The holy grail in protein folding research has always been to predict the tertiary structure of a protein given its primary sequence.  A similar but conceptually easier problem is to design a protein which will fold to a given structure with predicted secondary structure.  Many possible sequences could be designed to fold to the desired structure, which makes this problem easier compared to the folding of a given sequence to just one native state.  Kuhlman et al. have recently accomplished such a feat for a synthetic protein of 93 amino acids which they designed to fold to a unique topology not yet observed in nature.  This represents a significant advance over earlier attempts in which mimics of known proteins were made.   Such structures would be expected to fold in analogous fashions to the parent protein because of the necessary constraints placed by the need to fold to a compact state.

Chime Model: Top7 - A designed 93 amino acid protein with a novel fold

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