BIOCHEMISTRY - DR. JAKUBOWSKI
01/24/2010
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Learning Goals/Objectives for Chapter 2B: After class and this reading, students will be able to describe in general terms the procedures and chemical steps in the determinations of the following for proteins:
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As described in the Introduction to Proteins, we can understand proteins structure at varying level of complexity.
Figure: Protein Analysis from low to high resolution.
In the last chapter section, we learned about the charge and chemical reactivity properties of isolated amino acids and amino acids in proteins. The analysis of a whole protein is complicated since each different amino acid might be represented many times in the sequence. Each protein has an N-terminal and C-terminal amino acid and secondary structure. Some proteins exists biologically as multisubunit proteins, which adds to the complexity of the analyses since now the proteins would have multiple N- and C-terminal ends. In addition, isolated proteins might have chemically modifications (post-translational) which add to the functionalities of the proteins but also add to the complexiites of the analyses. To illustrate some of these issues, view the structure of the RhoA program below.
Jmol:
RhoA - a cytoplasmic protein - The complexity of protein analysis
Amino Acid Analysis (Composition)
At a low level of resolution, we can determine the amino acid composition of the protein by hydrolyzing the protein in 6 N HCl, 100oC, under vacuum for various time intervals. After removing the HCl, the hydrolyzate is applied to an ion-exchange or hydrophobic interaction column, and the amino acids eluted and quantitated with respect to known standards. A non naturally- occurring amino acid like norleucine is added in known amounts as an internal standard to monitor quantitative recovery during the reactions. The separated amino acids are often derivitized with ninhydrin or phenylisothiocyantate to facilitate their detection. The reaction is usually allowed to procedure for 24, 36, and 48 hours, since amino acids with OH (like ser) are destroyed. A time course allows the concentration of Ser at time t=0 to be extrapolated. Trp is also destroyed during the process. In addition, the amide links in the side chains of Gln and Asn are hydrolyzed to form Glu and Asp, respectively.
N- and C-Terminal Amino Acid Analysis
The amino acid composition does not give the sequence of the protein. The N-terminus of the protein can be determined by reacting the protein with fluorodinitrobenzene (FDNB) or dansyl chloride, which reacts with any free amine in the protein, including the epsilon amino group of lysine. The amino group of the protein is linked to the aromatic ring of the DNB through an amine and to the dansyl group by a sulfonamide, and are hence stable to hydrolysis. The protein is hydrolyzed in 6 N HCl, and the amino acids separated by TLC or HPLC. Two spots should result if the protein was a single chain, with some Lys residues. The labeled amino acid other than Lys is the N-terminal amino acid. The C-terminal amino acid can be determined by addition of carboxypeptidases, enzymes which cleave amino acids from the C-terminal. A time course must be done to see which amino acid is released first. N-terminal analysis can also be done as part of sequencing the entire protein as discussed below (Edman degradation reaction).
Analysis for Specific Amino Acids
Aromatic amino acids can be detected by their characteristic absorbance profiles. Amino acids with specific functional groups can be determined by chemical reactions with specific modifying groups, as shown in section 2A.
Figure: amino acid absorbance profiles
Amino Acid Sequence
Two methods exist to determine the entire sequence of a protein. In one, the protein is sequenced; in the other, the DNA encoding the protein is sequenced, from which the amino acid sequence can be derived. The actually protein can be sequenced by automated, sequential Edman Degradation.
Figure: Edman Degradation
In this technique, a protein adsorbed to a solid phase reacts with phenylisothiocyanate. An intramolecular cyclization and cleavage of the N-terminal amino acid results, which can be washed from the adsorbed protein and detected by HPLC analysis. The yields in this technique are close to 100%. However, with time, more chains accumulate in which an N-terminal amino acid has not been removed. If it is removed on the next step, two amino acids will elute, creating increasing "noise" in the elution step - i.e. more than 1 amino acid derivative will be detected. Hence the maximal length of the peptide which can be sequenced is about 50 amino acids. Most proteins are larger than that. Hence, before the protein can be sequenced, it must be cleaved with specific enzymes called endoproteases which cleave proteins after specific side chains. For example, trypsin cleaves proteins within a chain after Lys and Arg, while chymotrypsin cleaves after aromatic amino acids, like Trp, Tyr, and Phe. Chemical cleavage by small molecules can be used as well. Cyanogen bromide, CNBr, cleaves proteins after methionine side chains. The individual proteins must be cleaved using two different methods, and each peptide fragment isolated and sequenced. Then the order of the cleaved peptides with known sequence can be pieced together by comparing the peptide sequences obtained using different cleavage methods. Many proteins also have disulfide bonds connecting Cys side chains distial to each other in the polypeptide chain. Proteolytic or chemical cleavage of the protein would lead to the formation of a fragment containing two peptides linked by disulfides. Edman degration would release two amino acids from such fragments. To avoid this problem, the protein is oxidized with performic acid, which irreversibly oxidizes free Cys, or Cys-Cys disulfides to cysteic acid residues. A summary of the steps involved in protein sequencing are shown below:
PROTEIN SEQUENCING STRATEGY - 8 STEPS
PROTEIN SEQUENCING USING MASS SPECTROMETERY
A newer method of determining the sequence of a protein uses mass spectrometry. The following description is derived from: Corthals G.L., Gygi, S.P., Aebersold R. and Patterson, S.D., in Proteome research: 2D gel electrophoresis and detection methods, Ed. Rabilloud, T., Springer, New York, 1999, pp. 197-231.
"Biological mass spectrometry (MS) is now an indispensable tool for rapid protein and peptide structural analysis, and the widespread use of MS is a reflection of its ability to solve structural problems not readily or conclusively determined with conventional techniques. All mass spectrometers (MS) have three essential components that are required for measuring the mass of individual molecules that have been converted to gas-phase ions prior to detection. The components are an ion source, a mass analyzer and a detector.
Ions produced in the ion source are separated in the mass analyzer by their m/z ratio, and (usually) detected by a photomultiplier. MS data is recorded as "spectra" which displays ion intensity versus the m/z value. The two techniques that have become preferred methods for ionization of peptides and proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), due to their effective application to a wide range of proteins and peptides (Fenn et al., 1989; Karas & Hillenkamp, 1988).
Although different combinations of ionization techniques and mass analyzers exist, MALDI usually uses a time-of-light (TOF) tube as a mass analyzer while ESI is traditionally combined with quadrupole mass analyzers capable of tandem mass spectrometry (MS/MS). Instruments capable of MS/MS have the ability to select ions of a particular m/z ratio from a mixture of ions, to fragment selected ions by a process called collision induced dissociation (CID) and to record the precise masses of the resulting fragment ions. If this process is applied to the analysis a peptide ions, in principle the amino acid sequence of the peptide can be deduced. "
1/24/10:
In a typical MS/MS experiment, a protein is cleaved into protein fragments with
an enzyme such as trypsin, which cleaves on carboxyl side of positively charge
Lys and Arg side chains. The fragments are introduced in the MS,
where a peptide with a specific m/z ratio is further selected and fragmented by
collision with an inert agent. The figure below shows how peptides
typically fragment across the amide bond of the peptide backbone. In the
analysis of the resulting fragmentation peaks, peak y1 is determined which is
the C-terminal fragment of tryptic peptide (containing either a Lys or Arg side
chain). Peak y2, which differs by one amino acid in MW from y1, is
identified. All y fragments peaks have a common Lys terminal.
All b fragment peaks for a given peptide contain a common N terminal amino acid.
By identifying b and y peaks the actual sequence of the peptide can be
determined.
Figure: Peptide Fragmentation and Sequencing by MS/MS
Recently, MALDI mass spectroscopy has been used to sequence proteins obtained from fossils. Although most work on sequencing of fossil remains has centered on DNA analyses, some proteins, such as ostocalcin found in bone, is more stable than DNA, which is susceptible to degradation by ubiquitous enzymes. Osteocalcin from a 55,000 year old bison bone was identical to that from the modern bison., and was one amino acid different from the modern cow.
Conformational Analyses of Proteins.
A protein can be considered to have primary, secondary, tertiary, and quaternary structures.
Figure: Secondary Structure (purple - a helices, yellow - b strands. Image made with VMD)
Figure: tertiary structure (calmodulin - image made with VMD)
Figure: Quaternary structure (4 chains of hemoglobin - Image made with VMD)
2o Structure: The percent and type of secondary structure can be determined using circular dichroism (CD) spectroscopy. (The links below come from an animated tutorial on Electromagnetic Waves and Circular Dichroism by András Szilágyi ) In this method, right and left circularly polarized light illuminates a protein, which, since it is made of all L-amino acids, is chiral. (The mirror image would be a protein of the same sequence made of D-amino acids.) Circularly polarized light can be made when plane polarized light meet out of phase. If R and L circularly polarized light is passed through an optically inactive medium and if they have the same amplitude and frequency, the two waves combine (vectorially) to produce plane polarized light. Optical activity is observed only when the environment in which a transition occurs is asymmetric.
The peptide (amide) bond absorbs UV light in the range of 180 to 230 nm (far-UV range) so this region of the spectra give information about the protein backbone, and more specifically, the secondary structure of the protein. The main transitions are n --> p * at 220 nm and p --> p * at 190 nm. There is a little contribution from aromatic amino acid side chains but it is small given the large number of peptide bonds. The peptide bonds in a protein's asymmetric environment will absorb this range of light (promoting electrons to higher energy levels). In different secondary structures , the peptide bond electrons will absorb right and left circularly polarized light differently (for example, they have different molar absorptivities). Hence a, b and random coil structures all have distinguishable far UV CD spectra.
Stated in another way, if plane polarized light, which is a superposition of right and left circularly polarized light, passes through an asymmetic sample which absorbs right and left circularly polarized differently, then the light passing through the sample after vector addition of the right and left hand circularly polarized light gives elliptically polarized light. (Great link!)
If the chiral molecules also have a different index of refraction for R and L circularly polarized light, an added net effect is rotation of the angle of the ellipitically of the polarized light. The far-UV CD spectrum of the protein is sensitive to the main chain conformation. The CD spectra of alpha and beta secondary structure are shown in the figure below.
Figure: The CD Spectra of Alpha-Helix, Beta-Sheet, and Random Coils
Protein side chains also find themselves in such an asymmetric environment. If irradiated with circularly polarized UV light in the range of 250-300 nm (near UV), differential absorption of right and circularly polarized light by the aromatic amino acids (Tyr, Phe, Trp) and disulfide bonds occur and a near UV CD spectra result. If the near UV CD spectra of a protein is taken under two different sets of conditions, and the spectra differ, then it can be surmised that the environment of the side chains is different, and hence the proteins have somewhat different conformations. It will not give information about secondary structure of the backbone since that requires lower wavelengths for absorption of occur. Rather it can show differences in tertiary structure.
3o Structure: Clearly, the highest resolution understanding of protein structure requires a solution to the 3D structure of the protein. Once that is determined, it is easy to devise computer programs which will determine what part of the structure is in secondary structure. Three methods are presently useful to determine the 3D structures of proteins.
A. X-Ray crystallography: If crystals of the protein can be made, traditional x-ray crystallographic techniques can be used to solve the structure. X-rays irradiate a crystal, which scatters the x-rays, leading to constructive/destructive interference patterns. Using appropriate math, the interference pattern can be reconverted into the actual structure of the protein. Check out a fun example showing such a reconstruction of the Parthenon from its diffraction pattern!
These structures are not solution structures, but overwhelming evidence suggest that they do represent the solution structure. For instance, x-ray structure contain many water molecules which interact with each other and the protein, a finding expected for a structure that represents the solution structure of the protein. In addition, substrates and inhibitors can be infused into the crystal and bind with the protein, suggesting again a native-like structure for the protein in the crystal.
B. NMR: There are many protons in proteins which give a proton NMR spectra. The problem is one of assignment, since there are so many. Nuclei in different environments absorb energy at different resonant frequencies. When a proton spin flips, it goes to a higher energy state. It will return to the equilibrium state with some time delay.
Figure: 1D NMR spectra of a protein
If an unexcited proton is proximal in space, the magnetization can be transferred to the unexcited proton. This interaction is inversely proportional to the 6th power of the distance between them and is the basis of the Nuclear Overhauser Effect (NOE). A 2D NOE spectra shows peaks off the diagonal that are correlated, indicated that they are close in 3D space.
Figure: 2D NOSEY spectra of a protein
Multi-dimensional techniques, obtained when isotopes of N(15) and C(13) are present in the protein, can be used to actually obtain a 3D solution of a small protein. NMR and X-ray structure of the protein are almost superimposable.
Figure: NMR Structure of Proteins
Chime: Rubredoxin
from pyroccus furious: NMR and X-Ray Structure | Jmol
C. Homology Modeling: The structure of unknown proteins can be modeled theoretically if they have extensive sequence homology to another protein whose structure is known.
Of course, to characterize proteins rigorously, they must be purified from a solution containing many proteins. We will cover these techniques in the laboratory. Here is a quick summary of some of them:
Moodle
Online Quiz (PASSWORD PROTECTED):
PROTEIN I
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