Dr. Peter L. Davies, Queen's University, Department of Biochemistry
Queen's University Peter L. Davies
Professor of Biochemistry & Biology
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Lab Members:

Dr. Peter L. Davies
Peter Davies Yes, there is always lots of paper to push. My hard drive is equally messy but takes up less space and is easier to search. One 'piece of paper' I'm happy to have off my desk top is the recent review on ice-binding proteins that appeared last month in TiBS (doi:10.1016/j.tibs.2014.09.005). It has been 10 years since our last review on AFPs, and much has changed since then. One current writing challenge is submitting Stage 2 of the inaugural CIHR Foundation grant competition. Guess what I'm doing for the holidays? But come mid-January I'll be off on a mini-sabbatical leave to the University of Otago, Dunedin, NZ to work with Craig Marshall on new and exotic antifreeze projects. Thanks to 'Remote Desktop' it is easy to run the lab from anywhere. For example, my mug shot shows me working out of my west coast office! Lastly, a big shout-out for this year goes to Sakae Tsuda for organizing and hosting IBP2014 in Sapporo (IBP2014 website). It was a stimulating meeting and a great follow-up to IBP2011 that we hosted in Kingston (IBP2011 website).

Administrative Assistant:

Sandra Jimmo
My main responsibility it to keep Peter sane! This involves doing as much of the paperwork as possible, including assisting with grant applications, submitting papers and keeping the dozens of different "Common CVs" up to date. I prepare travel claims, cheque requisitions, and journals, keep track of the lab finances using FAST and PeopleSoft applications. I also help Peter make travel and accommodation arrangements for conferences, invited lectures and other work-related visits. Some of these things I also do for others in the Davies' Lab. They are exceptional people and I enjoy spending time with them all. Finally, when the boss and Sherry are both away it falls to me to water the plants in Peter's office -- like this Christmas cactus. Another big responsibility!

Research Assistants:

Sherry Gauthier
What does Sherry do? It might be easier to say what she doesn't do -- but here goes. She is den mother to the lab, guides newcomers through safety procedures, experimental protocols and optimal use of equipment. She keeps order in the lab, gives orders in the lab, and orders for the lab. She chides us when we leave a mess or use up the last of something without notifying her! Sherry is our collective memory of everything that was done in the past, of the registries and legacies left by former lab members and where everything is kept. There isn't a technique in the lab she hasn't done from the days when we used to do our own DNA sequencing and genomic libraries in lambda phage. Sherry breaks ground on new projects and pitches in when projects are stalled. When the cloning gets tough, the tough get cloning -- that is when we give it to Sherry. Here is a recent Southern blot she did with Laurie to probe the organization, evolution and amplification of type I antifreeze protein genes in fishes.

Research Associates:

Dr. Robert L. Campbell (website)
Robert Campbell My main interest is the study of protein structures by crystallography along with molecular modelling. One particular focus is in developing inhibitors of calpains. Calpains are a family of calcium-dependent proteases that function in a variety of normal roles involving limited cleavage of their protein targets under the control of the cellular calcium concentration. They are also implicated in a number of disease conditions that result from a loss of calcium homeostatis, such as heart attack, stroke and blunt trauma. The resulting increased calcium concentrations cause unregulated cleavage of many cellular proteins by calpains. We have determined the crystal structures of a number of complexes of small molecule inhibitors bound to the active site of calpain (shown superimposed in the image at right), but none of these are very specific. We are therefore designing new compounds that may prove to be specific inhibitors of calpain. We are using virtual screening (in silico docking) techniques along with molecular dynamics simulations to study possible inhibitors.
Dr. Laurie Graham
One strand running through my research has been the origins and evolution of antifreeze proteins (AFPs), which are a microcosm demonstrating all major evolutionary processes. Whether I am out collecting various insects during the winter or cloning the genes that produce these proteins, the goal of this work is to understand the diversity and similarities between the AFPs from various organisms. We found unexpected diversity in arthropods (insects and close relatives), where completely unrelated proteins have given rise to flat, repetitive faces that bind to the surface of ice. Sometimes, these ice-binding faces display a remarkable similarity (convergent evolution to a threonine-rich face), while other times they are different (alanine-rich). A variety of different beta-helices (right- or left-handed or flattened) have been co-opted, but for snow fleas, a completely different glycine-rich polyproline helix bundle was employed. This diversity suggests that antifreeze proteins arose independently in many branches of the arthropod tree. A similar situation is seen in fish, where four different AFP types (indicated by colour) are found.
Here we have four different types of fish producing highly similar alanine-rich alpha-helical proteins (red), but we have shown that the corresponding genes are not related. This is an extreme example of convergent evolution to a simple protein structure that is effective at preventing ice growth. In contrast, the type II AFPs (blue) arose from the same gene, which is remarkably similar in herring and smelt, considering that these fishes diverged over 200 million years ago. Even the non-coding introns are almost identical. We have shown that this AFP gene was transferred between these species by lateral gene transfer. Ongoing work continues to investigate the origins and evolution of AFP genes in various cold-tolerant species.
Dr. Qilu Ye
Calpain3 dimer Calpain-3 (CAPN3/p94), often called the skeletal muscle-specific calpain, is a multi-domain calcium-dependent cysteine protease whose function is not yet fully understood. A functional calpain-3 is indispensable for the maintenance of skeletal muscle. To date, there are more than 400 documented mutations in CAPN3 and its gene that lead to the development of limb-girdle muscular dystrophy type 2A (LGMD2A). Patients with LGMD2J and some tibial muscular dystrophy (TMD) also demonstrate a lower level of CAPN3. The whole enzyme is a homodimer of CAPN3, which undergoes very rapid autoproteolysis. It has two insertion sequences, IS1 and IS2, and a long N-terminal extension, NS, not found in other calpains. Our previous studies showed IS1contributes to the instability of calpain-3; probably by acting as a readily cleavable internal pro-peptide that occupies the active site of the protease core. IS2 is located just before the penta-EF-hand domain and is thought to bind to proteins of the sarcomere. I am using X-ray crystallography to investigate the structure and role of these two insertion sequences and ways in which calpain-3 can be stabilized.

Postdoctoral Fellows:

Graduate Students:

Tianjun Sun
Hi! I'm TSun. The goal of my research is to test the validity and universality of the 'anchored clathrate water' hypothesis as a mechanistic explanation for the irreversible binding of antifreeze proteins (AFPs) to ice. Winter flounder produces two different sized AFPs in the blood. The small isoform, referred to as type I AFP, is a 3-kDa monomeric alanine-rich α-helix. The large isoform, referred to as Maxi, is five times as long as type I and forms a homodimer. Otherwise, it is similar to the small isoform in terms of alanine richness (65%), helicity (>95% α helix), and 11- residue periodicity. Recently, we reported in Science the structure of Maxi determined by X-ray crystallography. Both 290 Å-long helix monomers fold exactly in the middle through 180º. In the dimer, the two hairpins are packed in a way that two N-terminal helices lie adjacent to each other in an antiparallel orientation, so do the two C-terminal helices. Surprisingly, unlike typical globular proteins, which have a dry protein interior, this four-helical bundle retains ~400 waters in its core. The internal waters are organized into two intersecting polypentagonal networks (see image to the right). For type I AFP, the ice-binding residues are located along one face of the helix. The ice-binding residues are also conserved in Maxi. However, they point inwards to bind and coordinate the internal water network. These interactions (mainly hydrogen bonding) are thought to stabilize the fold of Maxi, which has minimal protein contacts between helices. These ordered waters can extend outwards to the protein surface and likely are involved in ice binding. This study has implications in multiple disciplines such as the ice-binding mechanism of AFPs, the general protein folding mechanism and gas hydrate inhibition.
Koli Basu
Insects and other arthropods that are active in this area of Ontario in the winter months are likely to have antifreeze proteins (AFPs) to protect them from freezing. We test them for thermal hysteresis activity and this is how we discovered novel antifreezes in snow fleas, inch worms, and now midges (seen here on the windows of our lab). These tiny midges emerge by the billion in late April and early May to mate, lay eggs, and start another annual cycle. At this time of the year the adults can still encounter night frosts, which would be deadly for them if they did not have freeze protection. Purifying the natural antifreeze protein is relatively easy with repeated cycles of ice affinity purification. I have been able to derive the sequence of the new antifreeze protein and various isomers using tandem mass spectrometry and nucleic acid sequencing. Our lab has considerable expertise and past success in predicting 3-D protein structures from repetitive amino acid sequences. Sure enough I now have a convincing novel structure for the midge AFP and am committed to verifying this by structural biology methods. I will also look at surface bound waters on the ice-binding site to see if they are ice like and fit in with our current hypothesis for the mechanism of action of antifreeze proteins.
Shuaiqi Guo
Hi! I'm Phil. My thesis research is on bacterial adhesin proteins - a relatively new area for the lab. When we were studying the antifreeze activity of an Antarctic bacterium from an ice-covered lake, we realized that the ice-binding region (RIV) made up just 2% of a giant, extended protein (Fig. 1). This 1.5-MDa protein has the structural features of a bacterial adhesin. So, now we think the function of this protein is not to protect the host from freezing but rather to bind the bacterium to the underside of lake or sea ice in a zone where oxygen and nutrients are produced by photosynthetic organisms. I am using a 'dissect and build' approach to investigate the structure and function of all parts of the ice adhesin. Region II is a tandem series of ~120 immunoglobulin beta-sandwich domains that can extend the ice-binding domain up to 0.6 microns away from the bacterial surface. Region 1 is likely the attachment point to the bacterial outer membrane; and Region V might help secretion of the protein through the Type I secretion system. We know the least about the 80-kDa Region III - but not for long! Characterizing this giant ice-binding protein will help us understand bacterial adhesins in general, including those associated with human and animal pathogens.
Christian McCartney
Hi! I'm Christian. My research interest is the calcium-dependent cysteine protease calpain. Calpains are intracellular proteases found in all the tissue and cell types of the human body. One of our overarching goals in the Davies lab, is to design, test and implement calpain-specific inhibitors to prevent off-target proteolysis, which has been linked to diseases such as Alzheimer's, heart attack, MS, stroke, cancer and traumatic spinal cord/brain injury. Arguably, one of the most interesting and perplexing characteristics of calpain is its high calcium requirement -- multi-micromolar to sub-millimolar. This has prompted many to speculate as to how this protease becomes active within the cell, where resting calcium levels are maintained at multi-nanomolar concentrations. To address this question, several mechanisms have been put forward. We maintain that under physiological conditions, calcium concentrations in the microenvironment surrounding a calcium influx can temporarily achieve the amounts of calcium required to activate calpain without the need for extraneous factors or post-translational modification. To test our hypothesis we have synthesized and characterized a set of protein-based fluorogenic FRET reporter substrates incorporating the optimal calpain cleavage peptide PLFAAR substrate. This probe will allow us to distinguish between physiological and non-physiological calpain activation in the cell, and to determine when, where and under what circumstances calpain is activated. Furthermore, this will lead to the identification of relevant calpain substrates by comparative whole cell proteomics, and provide a cell-based system for testing novel calpain inhibitors that we are developing by structure-guided approaches...
Corey Stevens
My thesis projects involve protein engineering of ice-binding proteins to enhance or change their activities. In one project I am trying to make an antifreeze protein out of a flat-surfaced, structurally regular protein that has no ice-binding activity. Here the placement of appropriate side chains will be critical for organizing ice-like waters (which we can model) if these are to dock the protein to ice. In another project I am exploring the multimerization of an antifreeze protein as a novel reagent with enhanced ice-binding properties. We know that increasing the area of an ice-binding site or doubling the number of ice-binding sites can greatly increase the antifreeze activity of a construct. I want to explore the limits of this approach and deduce the basis for enhanced activity. The accompanying picture shows a model of many type III AFPs bound to a soluble support.
Tyler Vance
Tyler here. I work on bacterial adhesion proteins and their role in biofilms. A biofilm is a three-dimensional clustering of bacteria into a multi-cellular community that can withstand inhospitable environments, such as those induced by antibiotics. Biofilms are involved in approximately 80% of chronic bacterial infections and can hamper industrial processes and cause sanitation problems. Determining the molecular players involved in bacterial clustering may provide methods to combat these detrimental processes. With this goal in mind, our lab has begun to study bacterial adhesion proteins. In order to form a biofilm, microorganisms must be able to stick to a surface and to each other. Adhesion proteins provide this ability. Bacteria can use these proteinaceous appendages like tentacles, reaching out into the environment to grab and hold onto specific targets. We have identified adhesion proteins implicated in the formation of biofilms from several bacterial species, including the pathogen Pseudomonas aeruginosa and the oil-eating bacteria Marinobacter hydrocarbonoclasticus. We are now working to solve the three-dimensional structure of these proteins of interest, in order to tease out how they aid in the formation of biofilms and how these mechanisms can be potentially sabotaged.
Evan Andrin
I am interested in the structure-function relationships of ice-nucleation proteins (INPs). Despite having diametrically opposite functions, INPs resemble antifreeze proteins (AFPs) in many respects. Both INPs and AFPs contain similar ice-binding motifs, and the former has been modelled as a beta-solenoid structure similar to some of the most potent AFPs. My research goals are two-fold: First, I will elucidate what structural elements differentiate the function of INPs from AFPs. AFPs are generally small (< 10 kDa), soluble, single-domain proteins. Conversely, the INPs are large (~120 kDa), membrane-associated, and function as MDa-scale multimers. Second, using a 'dissect and build' approach I will determine the three-dimensional protein structure of the full-length ice-nucleation protein. Continuing research into INPs promises to improve the preparation and preservation of cold-storage pharmaceuticals, whole tissues, and frozen foods.
Sean Phippen
Hi there! My name is Sean, and I am working as a graduate student in the Peter Davies lab. Previously I worked with Phil on determining the structure of the membrane-associated region (called Region I) of a large bacterial ice-binding adhesin. Determining the structure of this region will hopefully shed light on the manner in which the adhesin is attached to the outer membrane of the bacterium. This project involved cloning, protein expression and purification, crystallization and diffraction data collection.
Currently, my work is focused on protein engineering nanomaterials with enhanced antifreeze activity. Attaching multiple antifreeze proteins to a scaffold results in an increased activity compared to single proteins (see, for example, Corey's project). This occurs because an antifreeze protein is more likely to bind to an ice crystal if it is attached -- through the scaffold -- to one that is already bound to the ice! My project involves exploring new scaffolds to attach these proteins to, and assessing the ability of the nanomaterials to reduce the freezing point of ice-containing solutions, to inhibit ice recrystallization or to initiate ice nucleation.

Undergraduate Students:

Brigid Conroy
Hi! I'm Brigid, going into 4th year of the Honours Biochemistry program here at Queen's. For my summer research project, I am working with Tyler Vance to investigate the three-dimensional structure of bacterial adhesion proteins that are implicated in the formation of biofilms. Some of these adhesins come from Gram-negative bacteria that are human pathogens. By understanding the structure-function relationships in these proteins we hope to find opportunities to block their ability to form resistant biofilms. The adhesion proteins are complex and contain many different domains that serve various roles. Using X-ray crystallography, I will attempt to determine the structure of those domains we think are involved in self-association that might serve to link the bacteria together.
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