BioMinds final blog

I had the opportunity to present the most recent results of my research work, supervised by Dr. Pastrana, at the BioMinds Research Day Poster Session.
During this activity, I visited three posters and interviewed those students involved in each respective project. Among these projects was that of M. A Ortiz, who studied the flavonoid content of medicinal plant extracts from several genus. Apparently, in several regions of Puerto Rico, islanders have popularized the use of some plants used as teas for their ‘’supposedly’’ high levels of antioxidants. Ortiz was interested in quantifying the antioxidant content of these medicinal plants, in particular their flavonoid content, to determine whether or not these plants could in fact provide the benefits many people allege.  Although some of the results were somewhat misleading, suggesting some of the tested plants contained more flavonoid than their overall antioxidant content, Ortiz pointed out that some of the plants they tested were complicating the quantification process and, consequently, more studies were necessary to obtain more trustworthy results.
Another poster I visited was that of T.K. Jessica, who studied the isolation and characterization of organic compounds from M. charantia. This plant is used in several regions of Puerto Rico for the treatment of Diabetes Type II. She was trying to isolate these compounds using size exclusion columns, and characterizing them using 1-D nuclear magnetic resonance. Relatively high concentrations of these compounds were needed in order to characterize them. Consequently, Jessica was using a mathematical approach to quantify the amount of sample she was able to recover using different solvents. Unfortunately, some of her calculations were not able to explain or predict all endeavors of sample recovery, predicting significant yields of sample when, in fact, low yields were recovered. By looking at the structure of analogous organic compounds to that which she’s interested in isolating, I noticed these are mainly ring structures that might potentially serve as chromophores. I suggested she could look more into this and try to use a different, more reliable analytical approach such as absorbance measurements of her sample.
Finally, I visited the poster of A. Lopez-Cruz, w ho worked under the supervision of Dr. C. Rinaldi from the ChemE department at UPRM. He worked on the preparation of magnetic nanoparticles for their use in biomedical applications such as cancer treatment. These nanoparticles contained covalently bound Chitosan, and were characterized using several approaches. I happened to be familiar with FT-IR, DLS and SEM data analysis, which were among the instruments he used to confirm the validity of his results. I’ve mostly used this sort of instrumentation for biophysic/structural biology  purposes, so it was interesting to see how other applications also benefit from this technology. I was mostly excited with this work for, although it is far from related to most of the work I perform, it was still the most related out of all three projects I came across with.

Mechanism of unfolding comes to light!

Based on the goals I proposed for my project this semester, approximately 50% have been completed. Not only have I been able to isolate the hsfi1 construct with up to ~75-80% purity, but I’ve also been able to conduct 2D-correlational spectroscopy studies for a hcentrin1 mutant, specifically E105K. These FT-IR correlation spectroscopy studies have allowed me to identify which secondary structures of the protein are perturbed and to what extent based on increasing thermal perturbations exerted on the sample. Furthermore, using spectral plots of these perturbations, I’ve been able to establish the sequential order of events that take place as the protein unfolds or destabilizes with increasing temperature. By comparing the sequential order of events of this mutant and that of other hcentrin mutants studied in the lab, we are able to compare the extent of stability of all wild type and mutant hcentrin proteins, with hopes of understanding the extent of instability different mutations bring to the protein. We hope to extend our 2D-COS studies to the understanding of hcentrin-hsfi1 complex formation.
Due to a number of factors previously explained in other blog posts, it’s been very difficult to fully isolate the hsfi1 construct. Nonetheless, as we’ve begun to understand the behavior of the protein more and more, we’ve modified the conditions of purification and have been very successful in moving the project forward. We have recently designed a slightly different modification of the purification protocol that we expect will greatly increase our chances of finally isolating the construct. This modification is based on the insolubility properties the protein presents, and its surprising affinity to the chromatography column used in comparison with the affinity tag ‘’GST.’’

BioMinds Poster Abstract

Conformational stability of hcen1 (E105K) upon thermal perturbation using two-dimensional correlational spectroscopy and hsfi1 construct isolation for subsequent dynamic characterization studies of hsfi1-hcen complexes
A. Castillo Rodriguez †, B. Pastrana †‡
† University of Puerto Rico, Mayaguez Campus
‡ Protein structural characterization center, chemistry department
research area: Biophysics and biochemistry
Centrins are a low molecular weight (~2000), subfamily within the EF-hand Calmodulin superfamily that play an essential role in centrosome duplication and contraction of centrin-based fiber systems. Although elucidation of centrin function and structure is yet to be fully explained, evidence shows interaction with other protein targets such as sfi1.
Sfi1 is a large protein mainly localized in the centrosome of higher eukaryotes. Its large sequence reveals 23 amino acid repeats in human proteins, separated by 10 residue linkers. Experiments have revealed the presence of a physical interaction between hsfi1 and centrin molecules, suggesting that each of the 23 amino acid repeats present in Sfi1 comprise a centrin-binding site. Nonetheless, very little is known of sfi1 proteins, and its structure is yet to be fully elucidated.
In order to probe conformationally sensitive delocalized states of Hcen1 E105K upon thermal perturbation and defeat spectral congestion, transient conformational changes were investigated using nonlinear Fourier transform infrared IR spectroscopy of the amide I’ vibrations and amide II’ bands in the mid IR region of the spectrum. Two-dimensional correlational spectroscopy (2DCOS) reveals a transient instability in secondary structure of the mutant protein as a function of increasing temperature. Backbone correlations confirm order of events, suggesting weak conformational perturbations in the loop regions initiate alpha-helical instability of the protein, thus promoting subsequent unsteadiness of the ß-strand composition. This latter event, in turn, finally initiates instability of calcium-binding sites of the protein, suggesting calcium ions are released just prior to Hcen1 E105K’s denaturation temperature.
Furthermore, the isolation of a highly insoluble human Sfi1 (hsfi1) D10 construct for future conformational dynamic studies with the mutant hcen1protein formerly described was performed using a GST affinity column. Purification of this construct has allowed us to endeavor co-isolation of hsfi1-hcen complexes and higher order hsfi1 domains using analogous purification methods.

BioBlog

This last semester, I hope to isolate the hsfi1 D10 construct for future dynamic studies, where we hope to gain a deeper understanding of the dynamics involved during complex formation of the hsfi1 construct with hcentrins. The isolation part of the project is nearly done. Nonetheless, there is great difficulty in maintaining the isolated construct soluble. Introduction of detergents to force solubility can interfere with our subsequent 2D-correlational spectroscopy studies. We hope hsfi1-hcentin complex formation will aid in solubility to the extent necessary for the dynamic studies we’re currently interested in conducting. We will also be performing 2D-COS studies for several hcentrin mutants using external temperature perturbations. We will use these studies to compare the order of events taking place during protein unfolding and see how each one varies with respect to the other. Analogous experiments will be conducted for the hsfi1-hcentrin complexes as soon as the hsfi1 constructs of interest are isolated.
As I had mentioned in the last time I posted, once the hsfi1 D10 construct is isolated and an official protocol is established, we will then endeavor purifying the hsfi1 D10-12 construct. The latter, as oppose to the former, is of utter importance, for it will allow us to follow the binding mechanism of, not only a single hcentrin with hsfi1, but possibly two hcentrins. Furthermore, we believe there might also we present an interaction between neighboring hcentrins once they bind to hsfi1. We hope 2D-correlational spectroscopy studies will allow us to confirm whether these interactions are present, and in which order of events  they’re taking place.

During this past semester…

During this past semester, I mostly worked with the isolation of hsfi1 domains, particularly hsfi1 D10, with hopes of successfully isolating this one. Success of this purification would serve useful for future isolation of the D10-12 construct, which is known to be a more hydrophobic dimer.  The problems we encounter throughout the isolation process ranged from domain solubilization to subsequent binding efficiency to the glutathione affinity column used, as well as optimal recovery of the pure domain. Unfortunately, our first attempts in obtaining soluble protein from our lysis procedure were not successful, primarily due to a poor protocol design. Because hsfi1 domains are highly insoluble, detergents are needed to aid in protein solubility. Sarcosyl is particularly used for this task. Unfortunately, Sarcosyl interferes with subsequent binding of the soluble protein once it is decanted to the affinity column. We were not aware of this at first. Triton X-100, a non-ionic detergent is used to precipitate Sarcosyl, forming large micelles that aid in Sarcosyl trapping, avoiding interference of Sarcosyl in the protein-binding step performed.  In summary, the addition of proper amounts of Triton-X100 detergent after Sarcosyl treatment did indeed aid in protein solubility immensely.
Although we have been unable to optimize our GST-tagged hsfi1 D10 domain affinity to the glutathione sepharose column used, we have reason to believe a DTT treatment in our lysis procedure, prior to the Sarcosyl treatment described above might potentially solve this issue. Future experiments are needed to confirm this hypothesis.
Several enzymatic cleavage assays were performed in order to estimate optimal amounts of protease needed for complete cleavage of our GST-tagged construct. These experiments were successful and are described in detail in the final semester report of the project.
We noticed that once the protease cleavage treatment was performed and the cleaved mixture was once again eluted through the column for GST-tag / cleaved construct separation, the amounts of recovered construct from the column were not comparable to those observed prior to this final column separation step.  We hypothesized that the construct could potentially have an affinity for the column matrix as well, even without its GST tag. This idea was suggested after acknowledging previous studies found in the scientific literature where hydrophobic constructs were found to bind the column matrix, as a consequence of inclusion bodies formed between them. Non-ionic detergent was used in some cases to aid in the solubility of the matrix/bound construct and subsequent elution.  We performed this step in our own experiment and did, indeed, recover cleaved protein bound to the column matrix, something that was not previously thought possible for our constructs of interest in the lab until now.

In conclusion, optimization in the binding activity of the construct will be sufficient to write an official purification protocol for this domain.  This one will be applied as well for the hsfiD10-12 construct.
Succesful isolation of this domain will allow us to use  infrared correlational spectroscopy techniques (2D-COS) to study the binding interactions of this domain with centrin proteins. We plan on performing these biophysical studies during the course of next semester.

Half way there…

  As far as the progress of my project, based on Augusts’ objectives, I believe it is reflected somewhere between 25-30%. I recently worked on a large scale purification of hsfi1 D(10-12). Although we had previous knowledge that this construct is fairly insoluble, we were not at all prepared for the intricacies involved in isolating it. Kilmartin et.al have isolated the construct previously and, in fact, published their own set of results using the same construct we are currently trying to purify. This group points out that a modified version of Frangioni and Neel’s GST-tagged protocol was used to increase the solubility of the construct, as well as its affinity and consequent isolation. We were able to perform a successful isolation of hsfi1 D(10) a few months ago. 

   Although the isolation of a single hsfi1 domain was a big accomplishment for us, it is critical that we purify both domains at once, in order to gain a deeper understanding of the dynamics involved in hsfi1-hcentrin binding. Nonetheless, even a single added domain, as we noted, can increase the insolubility of the construct dramatically, making it extremely difficult to purify and isolate. Even various consecutive lysis procedures in the protein pellet have not shown promising results. This past week we expressed the single hsfi1 D(10) construct, which we plan on purifying in large yields, in order to gain some insight into how this construct behaves, and move on the hsfi1 D(10-12) construct.

   We have also proposed using alternative methods for purifying these constructs, such as the use of a denaturing agent to increase the solubility of our-now- denatured protein, with hopes of renaturing the construct again once isolated. Nonetheless, not all constructs successfully fold back to their native state once denatured under certain conditions. A re-evaluation of the amino acid sequence of both constructs might prove beneficial, as sometimes adding or removing certain amino acids from the sequence being expressed can increase the construct solubility.

   In other news, I am still learning how to perform spectroscopic studies using 2D-correlational spectroscopy. I will perform a couple of dynamic studies using various hcentrin constructs we’ve isolated. Hopefully, once the hsfi1 construct has been isolated, we will then perform the same 2D-COS studies with the hsfi1-hcentrin complexes. That is really the goal of this big project. Understanding the dynamics of these proteins allow further understanding of those events involved during cell-division events.

If you bother, it will talk to you

During the past month, I’ve been meeting with Dr. Pastrana to discuss the theoretical background of two-dimensional spectroscopy (2D-COS), and its application to infrared. This instrument can provide a structural analysis of proteins under study, and can describe by means of a contour map representation, how their secondary structure is altered when subjected to different environmental perturbations. Although this technique has been used in conjunction with other spectroscopic techniques, our lab is currently interested in its use for 2D-IR spectral analyses.

Environmental perturbation studies can include pH gradients, temperature gradients, etc…
The way an experimental procedure is typically approached is described as follows: A series of perturbation-induced dynamic spectra are collected in a systematic manner, and then transformed into a set of 2D-correlation spectra by cross-correlation analysis (1). I look forward to using this instrument to test the hsfi1 domains I’ve been working on, as well as the hcentrin-hsfi1 complexes I’ve worked on as well. This data can then be compared with the circular dichroism experiments for comparison and/or supplemental structural characterization.
I’ve currently been working on the isolation of the hsfi1 domains 10-12 using a GST affinity column. Because we are trying to determine the most effective protocol purification for this protein, in conjunction with two more students, I’ve been purifying small protein pellet samples using variations in our current protocol, with hopes of determining which factors are contributing to the loss of large amounts of the protein throughout the several steps the purification procedure entails. I believe I’ve so far only completed 10 percent of what I’ve intended to work on for the semester. There is much to work on and learn from. Hsfi1 has shown to be very insoluble and difficult to handle. We expect to have a strong set of results

Summer’s gone

Throughout this past summer, I spent nine weeks conducting biostructural research under the guidance of Doctor Walter J. Chazin, from the biochemistry and biophysics’ faculty at Vanderbilt University, TN. His research interests seek in underlying molecular basis for biological specificity and biochemical funtion of proteins and nucleic acids using NMR spectroscopy, X-ray chrystallography, and other biophysical methods to characterize molecular structure and dynamics.

My project involved the binding affinity quantification of the 70N domain of Replication Protein A (RPA). RPA is the primary single-stranded DNA (ssDNA) binding protein in eukaryotes. It plays a central role in chromosomal DNA replication, repair and recombination pathways, protecting ssDNA from degradation by nucleases.  RPA also mediates interactions with specific proteins active in these various DNA processing events. This multi-functionality correlates with RPA’s modular structure.

RPA has three subunits, each named after its molecular weight:  RPA70 (domains N, A, B and C), RPA32 (domains N, D and C) and RPA14 (single domain).  The N-terminal domain of RPA70 (70N) is flexibly linked by an 80 amino acid linker to the rest of RPA 70.  It has long been established that RPA binds ssDNA with nM affinity through the action of domains 70A, 70B, 70C and 32D using 3 modes of binding. Tandem DNA binding by domains 70A and 70B  is required for high affinity.

Recently, a proposal has been made that 70N contributes to DNA binding function.  However, 70N binding affinity is more than 1000-fold weaker than RPA70AB and all evidence shows 70N is primarily a protein-protein interaction domain targeting transcription factors and checkpoint proteins such as p53 and ATRIP, respectively.  This study aimed to resolve this controversy by analyzing the effect of 70N on the ssDNA binding activity of the high affinity RPA DNA binding domains, 70A and 70B. We proposed the use of size exclusion chromatography, dynamic light scattering (DLS), and isothermal titration calorimetry (ITC) to do a systematic comparison of the DNA binding properties of 70AB versus a 70NAB construct. We expect to show that 70AB DNA binding affinity is the same as 70NAB. This will provide conclusive evidence that the 70N domain is not involved in binding ssDNA.

For the course of this academic term, I will be in charge of isolating several domains of the hsfi1 protein. We have previously endeavored purifying this protein using several chromatographic techniques. After many unsuccessful trials using nickel columns for histidine-tagged hsfi1 proteins, we were finally able to isolate and sequence the hsfi1 protein. This isolated protein was a GST-tagged hsfi1 construct. Nonetheless, we were never able to isolate hsfi1 again. A recent, successful expression of both, hcentrin and hsfi1 proteins were obtained from a single protein batch pellet . It is thought hcentrin protein aids in hsfi1 solubility. We attribute our previous unsuccessful attempts in purifying hsfi1 to the single over expression of this protein absent of hcentrin. We believe the recovery of hsfi1 following the same previous protocols used for its purification will yield larger ammounts of the protein.  I also hope to repeat my circuar dichroism experiments of my hsfi1 and hcentrin constructs and complexes for secondary structure characterization. Due to my previous summer experience using ITC instruments for thermodynamic characterization of protein complexes formed in solution, I also hope to quantify the binding affinities of hcentrin and hsfi1 proteins.  One of my goals for this term and the next would be to  crystallize the hcentrin-hsfi1 complex. It’s been difficult to crystallize either one of these isolated proteins. Structural stability of the hcentrin-hsfi1 protein complex should yield more promising crystals that can diffract and allow its structural topology to be characterized.

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The end of a start

It’s been quite a semester, both inside and outside the lab. For starters, I am finally working directly with Prof. Belinda Pastrana. Before then, I was working directly with one of her research lab technicians. Nonetheless, because I was working mostly in the area of protein expression and purification, I felt like my interests were more geared towards biophysical research studies, and thought: ” Who better to work with than the woman who knows biophysics best?” And so now our arguments concern spectra analyses of all sorts, a lot of planning, calculations…the usual science talk; it’s great!
I have to say I’ve managed to learn more than I expected this term. I, for one, feel like I can sit down and think critically about things, and even when certain approaches I take are not fully supported by convincing, explicit arguments or data, it always manages to work through right at the very last end…its all a matter of knowing why you approach things a certain way, even when they might not make that much sense at that very momment. I can sit down and read a research paper from cover to cover, and actually develop a full interest in it as I read through the lines. Even if the research isn’t directly concerned with my current research interests. Research overall always seems to have a purpose, and allowing yourself to appreciate the science that’s being worked on all around you is something I used to take very much for granted. Plus, I can’t help but enjoy questioning my mentor about things when we have different oppinions over a certain subject. It helps in reminding me that I’m not alone in this; that we are two different minds working on the exact same thing. We must be up to something big!
I think my biggest challenge overall this term was to move from that “learning phase” we all go through everytime we’re welcomed to a lab for the first time, and finally feel like I’m one of the big boys. Prof. Pastrana likes making sure students are there for their love of research, not just class credits and recommendation letters; I finally got around to convincing her. No one would spend (sometimes close to 20-24) hours a week in a lab for nothing. Ok, so I don’t spend peak hours at night in the lab anymore but lets be realistic, very few people at the undergraduate college level are that commited to research.
I hope this next term can be much more busy. I see many drastic changes currently taking place at the lab, which makes things much more fun and interesting to look forward to. Hopefully we’ll get a paper out soon on a protein complex Pastrana has been working on with us students. For the time being, I will finish up (hopefully) with my CD spectrana analysis for this term, and will focus on other aspects of research currently taking place at the lab as we speak. I will be working under the guidance of Doctor Walter Chazin from the structural biology department at Vanderbilt this summer. I’ve heard he’s an amazing mentor, has many facilities in his lab, ranging from mass spec to NMR (and he was the student of a Nobel prize ). More on my summer research soon to come. This will be a summer!

Getting techie with it

In order to succesfully execute research experiments, one must fully “get” how lab instruments are used. This is, to a certain extent, why some of us spend a large scope of our first term doing research learning basic lab techniques (thank God that phase is long gone now). I had my fair fun making gels for protein runs (electrophoresis), and am now experiencing the more technical side of my research. Like I mentioned in my previous post, I’ve been working with circular dichroism, and should soon be using SELCON3, which is a FORTRAN ran program for CD spectra analysis. In a nutshell, I can get a good estimate of the structural conformation of those proteins analyzed. Some alpha helix here, some beta sheet there…random coil anyone?
If the structure of my protein mostly random coil, will the random coil composition change to a large extent once it interacts with another protein? These things, SELCON3 can tell me.

I think one of the most difficult things I’ve had to work on is getting the actual concentrations of my protein solutions right. We originally went for a 4% microgram/microliter concentration, but noticed by our spectra analysis that we simply needed a higher concentration. So now we’re going for 16%.

Might sound simple, but these calculations require a precise calibration curve of those proteins tested, which calls for spectrophotometric analyses of samples under a range of concentrations. In short, different concentrations absorb different (UV rays that is), and if you’re good with the pipette, you have yourself a very straight calibration curve, with a slope that approximates the epsilon value, an a correlation coefficient of 1, or 0.9999…..

Can’t argue, anything is better than making SDS gels.

I recently had the opportunity to visit 3 blogs. I was surprised to find out one of the students was also working with a proteins (specifically FAP1 yeast protein). His group is also interested in protein structure studies, and they’re but a few lab rooms away.
Another student is working with syntheses of quiral alcohols for neurodegenerative studies. I’m quite ignorant in the field, so I’m pretty excited to know why the interest in these particular alcohols over other alcohols in general.
Last, a student that is using the mortality of shrimp and tropical plant extracts from Puerto Rico to study antitumor properties from these plants. I guess mice needed a small break
I wonder if tropical Puerto Rican plants have ever shown anti tumor properties previously? Could this be the reason why this group has develop an interest in those potential properties tropical plants possibly share? What could tropical plants share in common that other plants don’t, that has scientists in a search for antitumor properties among these? Hopefully I understood the scope of their research project.

It’s been quite an adventure

Bis nachste woche !
Ich habe manchmal Hausaufgabe

And what is it you do again?

You “Great, so you work with proteins…you and a million other scientists.”
I “Considering the number of proteins out there, that’s a nice rough estimate. You should major in statistics”
I ” Before you reply, did you know a major in statistics is, by far, one of the least popular majors around? I heard this from an MIT student.”

You “…”

So what is it I do again?

Simple!

Centrin, a calcium binding protein, regulated by phosphorylation and calcium is found to be essential in centrosomal duplication during cell division. Centrin undergoes structural changes that occur upon phosphorylation of the protein and its subsequent interaction with other proteins such as Sfi1.

So what’s the overall importance of this fact?

Well, it turns out that hcentrin proteins (short for human centrin) have been found to manifest on a larger scale during cell division of non-viable cells (cancerous cells in this particular case).
So why do we see such an increase in the number of hcentrin proteins, as compared to the small amount present for viable cell division?

Good question…
We do not know, but we’re terribly excited to find out!
Where do we start from though?

Well, Our research is primarily focused on protein structure and dynamic studies. We wish to perform biophysical analyses on our proteins of interest in order to elucidate the interactions these have with other proteins such as hsfi1, which we’ve found interacts with hcentrin. But besides understanding how this interaction takes place, we’re also interested in observing those conformational changes these proteins undertake. This could serve useful for future drug design experiments, as a reference tool for other centrosomal proteins studies, etc…

Convincing, maybe?
I’ll endeavor to by the end of this year

I’ve recently been purifying hsfi1 protein pellet samples that are expressed in the lab. For this particular protein I use an affinity column. The idea behind this procedure consists of the following:

The protein is expressed in a fermentor (you know…you take the vector [plasmid] and you insert that gene of interest you wish your E. Coli cell to express. In this case, it would obviously be hsfi1. Once the cell density increases inside the fermentor, and a satisfying amount of protein is estimated to have been expressed, these pellets end up in my hands and I take care of actually digging inside these pellets on a hunt for protein. But wait! These proteins in particular are GST tagged (this term will make sense in a minute). Consequently, when we transfer our sonicated pellet through the affinity column, this one contains a matrix which active composition I cannot recall at the moment, that binds GST tagged hsfi1 to the column. It separates it from the rest of the protein soup, none of it which contains any GST and can not therefore bind to the affinity column (remember this protein came right our of a cell, filled with everything from a golgi apparatus, to an endoplasmic reticulum, lipids, etc…multiply all those organs by a couple of thousands, a large number of proteins, and quite a bigger number of everything else a cell could possibly hold, and you got yourself quite a job). Once separated, you will most likely use other purification methods that might include other chromatographic columns (must not forget to cut that GST tag out of the protein once done with it..hooray for enzymes!).

Anything else?

I’ve recently been working with two synthetic hsfi1 protein domains (p10, p21). I’m currently using circular dichroism to measure their optical activity (in other words, an analysis of their secondary structure). By measuring them independently, and also interacting with centrin (protein-protein complexes), I can see any structural changes from either protein (random coiled, alfa helix, beta sheet, etc…) We also use calorimeters (DSC, ITC), Infra-red, etc. I have yet to work with these other instruments though. I believe I might start with DSC and infrared shortly. We shall see.

I think I work at an amazing lab. The research we do is absolutely incredible! I’ll let you in a little on protein cristallyzation (did I spell that right?) at our lab soon. A group is non-existent without its leader. I think I did the right thing in choosing my leader (Prof. Belinda Pastrana).