The Aldehyde Class Of Crosslinkers

Published Date: 02 Nov 2017

Answer. Once an unknown protein has been retrieved from protein pull-down experiments, the next logical step would then be to determine its identity. One of the ways by which this can be done is through mass spectrometry. This method is commonly used to explore the composition of biomolecules or chemical compounds. One of the more popular types of mass spectrometry utilized at present to analyze unknown proteins is called MALDI-TOF. Its popularity stems from the ability to handle larger and heavier biomolecules without fragmenting them during the ionization process. While the general idea behind the process is found in other conventional techniques like Fast-Atom-Bombardment (FAB), Secondary-Ion MS (SIMS), and others, MALDI-TOF takes a more sensitive and unique approach.

First, the protein sample needs to be dissolved in an appropriate solvent. Typically proteins can be dissolved in water or a polar solvent such as acetonitrile. Acetonitrile is more commonly used because of its low viscosity compared to other polar solvents such as DMSO1. The particular reason here is more mechanical in nature as the highly viscous DMSO will clog up the tubing in the mass spectrometry machine. The next step is then to add a matrix to the dissolved protein solution. Two particular characteristics of these matrices are acidity and strong UV-absorption capability. Acidic matrices are chosen to help promote ionization by providing a proton source, and quick UV absorption is also necessary in order to absorb the irradiation from the MALDI-TOF laser1. Once the unknown protein sample has been dissolved in the appropriate solvent and matrix, it is then spotted onto a MALDI plate. Here, a laser is then fired at the sample essentially vaporizing it into matrix crystals with the unknown protein embedded into them. The energy that is absorbed from the laser by the matrix leads to its ionization1. Since the protein sample has been co-crystallized with the matrix after the vaporization, protons from the matrix are then transferred to the protein resulting in their ionization. The energy transfer results in a desorption of the protein from the matrix into a gas state1. It then travels through an electric field that acts as an ion accelerator toward the mass analyzer or detector. The particular principle behind this is why it has been termed TOF or Time-of-Flight. Since the charges of the ionized molecules are the same as well as their energy, the only different characteristic is their mass. Thus, lighter molecules will reach the detector first, as their speed will be greater and vice versa. Consequently, what the detector is actually measuring the time of flight of each ion it receives. This data then provides a spectrum using the TOF of each ion and translating that essentially to mass and thereby amino acid identification. This can be applied to protein fragments in the exact same manner. The MS spectrum that results from detection provides a series of peaks of particular molecular weights determined by MALDI-TOF on a mass-charge ratio. The sequence of the protein fragment can then be determined by comparing the mass difference between the peaks1. However, in order to identify an unknown protein from MALDI-TOF, typically, the protein sample is digested by a protease such as trypsin or chymotrypsin to produce fragments of unique size. This digested sample is then run through MALDI-TOF MS to provide peaks in the spectrum that represent cleavage products1. The molecular weight of the peaks can be searched for via database to determine the identity of the protein based on its "fingerprint" in a technique known as peptide mass fingerprinting2.

2. There are several techniques to measure intracellular free Calcium concentrations. Describe methods to measure Ca2+ using fluorescence in different compartments within a cell, for example the mitochondria, endoplasmic reticulum and the cytoplasm.

Answer. Eukaryotic cells are very compartmentalized. As such, proteins require a signal sequence that targets them specifically to the compartments within the cell for which they are destined. For example, proteins targeted to peroxisomes have a distinct carboxy-terminal signal sequence3. Cleaving this sequence essentially eliminates the ability of the protein to target itself to peroxisomes. Likewise, adding this signal sequence to a random protein would result in its import into the peroxisome3. Therefore, this knowledge can be applied to measuring intracellular Calcium concentrations in different compartments. There exists Calcium fluorescent probes that essentially bind to Calcium inside the cell and fluoresce causing a shift in intensity that can be quantified and correlated with intracellular Calcium concentration. Typical Calcium concentration assays only seek to determine the total Calcium concentration within the cell. Thus, in these assays the cell is incubated in the fluorescent dye allowing it to be absorbed. The assay then calls for the treatment of the cells with a specific ligand that trigger intracellular Calcium release from cellular compartments for the dye to bind within the cytosol. However, if we wish to determine only the concentration of Calcium within specific compartments in the cell such as the mitochondria, endoplasmic reticulum, and the cytoplasm, then the fluorescent probes must be targeted to those compartments. Therefore, Calcium fluorescent probes can be cross-linked to the signal targeting sequence of the compartment you wish to assay for Calcium concentration. For example, to target the probes to the mitochondria, they need to be cross-linked with the amino-terminal targeting sequence found on all proteins targeting to the mitochondrion. Likewise, the endoplasmic reticulum requires a signal sequence at the N-terminus composed primarily of hydrophobic amino acids to target to the ER and be imported inside. However, if we wished only to measure the Calcium concentration within the cytoplasm, then we would use only the fluorescent probe itself, unaltered so that it is targeted to none of the compartments.

3. Describe the mechanisms for insertion of a protein into the cytoplasmic membrane and the outer membrane of a Gram-negative bacterial cell. What are the signals carried by a protein that are required for its integration into each membrane?

Answer4. Gram-negative bacteria have a series of assembly machineries that serve to insert membrane proteins into either the inner or outer membrane. In the case of inner membrane proteins, there exists a signal recognition particle (SRP) composed of an Ffh protein in conjunction with a 4.5S RNA that associates itself with a nascent polypeptide chains emerging from ribosomal complexes5. This SRP serves to target the growing polypeptide chain to the SRP receptor, FtsY5. GTP hydrolysis of this SRP-FtsY complex then results in a translocation of the nascent polypeptide into the SecYEG general secretory complex. This machine is composed of SecYEG, the SecA ATPase, and SecDFyajC. All three seem to promote the efficient folding and insertion of integral membrane proteins into the inner membrane. The process for this can proceed through two different ways. In the first mechanism, the SecYEG translocase functions to insert transmembrane segments of the growing polypeptide chain laterally into the inner membrane4, 5, 6. Alternatively, the transmembrane segments can be transferred directly over to the YidC insertase for folding into the inner membrane4. The YidC insertase like the SecYEG complex is also known to participate in the folding and insertion of proteins destined for the inner membrane. However, insertion by YidC typically proceeds through a SecYEG translocase independent manner4. Unfortunately, the mechanism by which proteins destined for the inner membrane are targeted to YidC is still largely unknown5. Distinct to inner membrane proteins however, is the presence of an anchor that keeps the protein embedded in the inner membrane preventing it from translocating elsewhere6. This anchor is present in the form of very hydrophobic alpha helices.

However, proteins that are destined for the outer membrane proceed through a longer process. Whereas integral membrane proteins are mostly composed of hydrophobic alpha helices that keep them anchored to the inner membrane, outer membrane proteins are composed of very amphipathic beta strands. The lateral gate in the SecYEG translocase opens only when large hydrophobic residues are translocated into the complex. Thus, when these amphipathic OMPs are targeted to the SecYEG translocase, they continue to be translocated across the inner membrane and into the periplasmic space6. It is also important to note that OMPs are targeted to the SecYEG translocase via an N-terminal signal sequence in conjunction with the SecB chaperone8. Once these OMPs reach the periplasm, the chaperones SurA and Skp associate with the polypeptide chain to prevent aggregation and misfolding9. It is then targeted to the Beta-Barrel Assembly Machinery Complex (BAM) by what is believed to be a particular signal sequence present at the C-terminus of the polypeptide chain7. At this BAM complex, SurA assists BAM in catalyzing the assembly of the OMP for insertion into the outer membrane. While the mechanism of OMP insertion via the BAM complex is still largely under debate, a recent model suggests that OMP polypeptide interaction with the Polypeptide-transport-Associated (POTRA) domains of the BAM complex trigger folding of the beta barrel with the aid of SurA leading to the insertion of the OMP into the outer membrane7, 10.

4. What are the different classes of chemical crosslinkers used for stabilizing a protein – protein interaction in bacteria? Provide an example of each class and determine whether the crosslinker of interest is suitable for crosslinking two interacting proteins in the cytoplasm or the cell envelope (inner and outer membranes and periplasm) of a Gram-negative cell. Describe the methods available to break the crosslinking without destroying the targeted proteins, what amino acids can be targeted and what chemistries are involved.

Answer11, 13. Crosslinkers are essentially molecules that can join two other bio-molecules/compounds of interest together by attaching their ends to specific functional groups. Because the interaction is so specific, there are several classes of crosslinkers based on reactivity groups. They are as follows: Carboxyl-Amine, Amine, Sulfhydryl, Aldehyde, and Photoreactive. The Carboxy to Amine reactivity class of crosslinkers, as the name implies, couples carboxyl groups on the proteins of interest to amine groups. For example, EDC or EDAC functions to interact with the carboxyl group on one protein to form an amine reacting intermediate that can then form an amide bond with an amine group on the other protein of interest. EDC is not particularly suitable for crosslinking two interacting proteins of interest in the cytoplasm or the cell envelope because proteins typically contain many carboxyl and amine groups for which EDC can react with. The result would be a random crosslinking between polypeptides with not very much specificity at all. Unfortunately, this crosslink is not cleavable and so the proteins of interest cannot be recovered individually.

NHS esters represent the Amine reactive class of crosslinkers. They are similar to EDC in that they react with only amine groups to form a crosslink, however, they do not interact and associate carboxyl groups to amine groups. Sulfo-NHS esters in particular are not suitable for crosslinking proteins in the cytoplasm or cell envelope because of its charged group preventing it from crossing the membrane. However, DSG is can permeate the membrane and target the amine groups of proteins in the cell envelope and cytoplasm. And to recover the proteins individually, while DSG is not cleavable, DSP, which also crosslinks amine groups, contains a disulfide bond that is cleavable by a thiol such as DTT.

Pirydyl disulfides are grouped under the Sulfhydryl class of crosslinkers. SPDP is an example from this class and serves to form a crosslink, in this case a disulfide bond, between an amine group and sulfhydryl group. SPDP, however, cannot permeate the membrane. Consequently, it is not suitable for crosslinking interacting proteins in the cytoplasm or inner membrane. Although, it can crosslink two interacting proteins found on the surface of the outermembrane. The link between your two proteins can then be cleaved through a reduction of the disulfide bond using a reducing agent such as DTT.

The Aldehyde class of crosslinkers is populated by hydrazides and alkoxyamines. The hydrazide, Sodium meta-Periodate, converts cis-glycols to aldehyde groups that can then form a crosslink at a pH range of 5-7. This is a very common crosslinker to use if your two proteins of interest are glycoproteins. Thus, since glycoproteins are typically found in the cell envelope, this crosslinker is more suitable for crosslinking glycoproteins in the cell membrane than in the cytoplasm. SDA is an example of the photoreactive class of crosslinkers. It conjugates amine groups to any other functional group when activated by UV light. It also comes in charged and uncharged forms to target proteins specifically on the cell surface or cytoplasm respectively.

There are a few methods available for breaking crosslinks. However, they are very dependent upon the crosslinker that is chosen, as some simply are not cleavable. Crosslinkers containg a disulfide bond can be targeted for cleavage by a thiol such as DTT (Hermanson). Those with an ester group can be cleaved by hydroxylamine at more basic pHs. A crosslinker with a diol group can be cleaved through oxidation with sodium periodate. The last method concerns crosslinkers with a sulfone group as it is cleavable by the addition of a base.

5. What types of detergents are useful for solubilizing bacterial inner and outer membranes? Provide examples of the various classes of detergents and describe their advantages/limitations.

Answer12. When choosing a detergent for solubilizing the proteins from the inner or outer membranes of bacteria, it is important to utilize a detergent that would not alter the structure or function of the protein(s) of interest in any way. This is especially critical in protein purification and crystallography as the integrity of the protein must remain intact. There are several classes of detergents that are typically used for solubilizing membrane proteins. The first is the class of non-ionic detergents also known as polyoxyethylenes. These uncharged detergents have typically been categorized as one of the milder detergents that can be used, as they have little to no impact on protein structure and function. Some of these non-ionic detergents are composed of particularly short hydrocarbon chains. This has been found to affect the function of proteins solubilized by the detergent much more so than detergents consisting of longer chains. Thus the longer chained Beta-octyl-glucoside non-ionic detergent, for example, is more commonly used as in the solubilization of membrane proteins. Thus, this mild class of detergents provides a good choice for usage in the extraction of proteins from membranes for which the function of the protein must remain unaltered.

The second class of detergents belongs to the zwitterionic detergents. This class of detergents is typically more damaging to protein function than non-ionic detergents. However, they represent the middle ground between the three detergent classes. CHAPS, for example, is a very commonly used zwitterionic detergent. While this class of detergents does cause more denaturation, it allows you to isolate out proteins that may be complexed very well. The reason for this is that zwitterionic detergents like CHAPS essentially prevent protein-protein interaction. The trade-off once again, however, is at a cost to protein function.

The third class of detergents is perhaps the most damaging. Ionic detergents such as SDS are almost never used for protein extraction from membranes. This is very much true if you wish to retain protein function, as SDS will simply denature the protein. The advantage of ionic detergents such as SDS however is rather unique. Studies found that ionic detergents are very efficient as solubilizing hydrophic alpha helices. Thus, ionic detergents may possibly be of use for the extraction of proteins found within the inner membrane of gram-negative bacterial cells. The reason for this stems from the fact that proteins that are inserted into the inner membrane of gram-negative bacteria by SecYEG/YidC typically consist of very hydrophobic alpha helices.

6. Compare and contrast the different types of molecular machineries used for the secretion and assembly of pili on the surface of bacterial cells. Make sure to include minimally P pili, type 1 pili, type IV pili, conjugative pili and pili from Gram-positive bacteria. Also, provide a short (1-2 sentences) description of the processes in which these different pili are used.

Answer. There are various different types of molecular machineries utilized in the secretion and assemblage of pili on the bacterial cell surface. Uropathogenic E. coli (UPEC), for example, are known to express P pili on their cell surface14. They are assembled via a chaperone/usher pathway whose components are expressed by the pap gene cluster15. This pili secretion system utilizes chaperones to translocate pilus subunits stably to the usher in the outermembrane. This usher can then catalyze the assembly of the pili onto the surface of the cell. These P pili are critical for the virulence of UPEC as these thin and rigid, but nonflagellar-like, protrusions from the bacterial surface are responsible for mediating bacterial adhesion to host cells. P pili in particular target specifically to galactose of glycolipids present on the epithelial cells lining the urinary tract16. The resulting colonization of UPEC following P pili adhesion results in urinary tract infections. Should the UPEC migrate up the urinary tract into the kidney, pyelonephritis may result. The fim gene cluster also comprises another chaperone/usher pathway and its own unique pili called the type 1 pili15. These pili also form a thin and rigid hair-like appendage that serves to mediate bacterial adhesion to the host cell surface. However, they are distinctly different from P pili structurally as seen by their shorter and less flexible fibrillum tip. In the case of type 1 pili, they target specifically to mannose moieties of glycoproteins found on bladder cells. Adhesion to bladder cells by type 1 pili then allows UPEC to invade the bladder resulting in cystitis16.

Type IV pili are much more diverse in function compared to P and type 1 pili. In addition to mediating bacterial adhesion to host cells as well as other bacterial cells, type IV pili are also implicated but not limited to the formation of biofilms, DNA uptake, and twitching motility, which is flagella-independent movement of the bacterium16, 17. Whereas type 1 pili measure approximately 10nm in length, type IV pili reach lengths of several micrometers. In addition to this, they are uniquely able to extend and adhere to surfaces before pulling themselves closer by retracting the pili. In the case of N. meningitidis, the type IV pili allows for binding to host cells in the nasopharynx. This allows the colonization of the bacterium where these very same pili can facilitate the formation of micro colonies16. These colonies of bacterial cells can invade the host epithelium and gain access to the blood stream causing bacterial meningitis. Unlike the type 1 and P pili, however, the genes required for the assembly and secretion of type IV pili do not originate from any single gene cluster. Unfortunately the molecular details for the assembly of these pili have yet to be fully elucidated. Its secretion and assemblage, however, is thought to occur in a manner similar to that of the Type II secretion system16. This particular system is composed of a basal body located in the inner membrane that anchors the translocator in the outer membrane. It is from the region of this basal body that type IV pili subunits are thought to be incorporated into the growing pilus18.

The type IV secretion system is most similar to the system used by conjugative pili for assembly and secretion to the surface19. A translocation pore located in the outer membrane associates itself with an inner membrane imbedded protein that spans the periplasm. Within this complex is where pili subunits form a hollow conjugative pilus that produces an opening in the cytoplasm to the tip of the pilus in the extracellular milieu. These pili can be just as long as type IV pili if not longer and are responsible for enabling a bacterial cell to engage in DNA transfer with another bacterial cell19. Pili in gram-positive bacterial cells are rather different. While, gram-negative cells have translocation pores in their outermembranes to assist in the assembly and secretion of pili, gram-positive bacteria have no such membrane or pore. Pili subunits are secreted across the membrane in gram-positive bacteria by the SecYEG translocase where they polymerize on top of sortases that are imbedded in the membrane. The sortase can then transfer the polymerized pilus to lipid II precursors that facilitate the anchoring of the pilus to the cell wall20. Like the p, type 1, and type IV pili, gram-positive pili are also responsible for mediating bacterial cell adhesion to host cells as well as aiding in the formation of biofilms. Unique to these pili, however, is the ability to provoke the inflammatory response by host immune defenses. This can then enable the bacterium to invade host tissues20.