Alpha Crystallin Structure and Function

The Alpha Crystallins are ~20 kDa small heat shock proteins (sHSP) that play several important roles in the eye.  There are two expressed forms of Alpha Crystallin; Alpha A Crystallin (AAC) and Alpha B Crystallin B (ABC). Our lab will be focusing on ABC.  This protein is well conserved and plays two important and overlapping roles. First, it is a small heat shock protein that prevents misfolded proteins from aggregating in the lens of the eye. This delays or prevents the formation of cataracts, the number one cause of blindness in the world.  Second, the protein takes part in a diverse oligomerization with other crystallins in the lens. This oligomerization is mediated by several protein-protein interaction surfaces in alpha crystallin. The Eisenberg Lab recently solved the crystal structure of most of this protein that showed these interation surfaces, and hinted at implications of its domain swapped C-terminal strand.

With leadership from post-doc Dr. Jim Hebda, our lab has focused on two important questions. First, what is the mechanism of chaperone function. To this end we will study model aggregation systems such as the insulin B-chain. Second, how does each of protein--protein interface contribute to oligomer stability and chaperone function. We will study the oligomerization of the alpha crystallins and the importance of each of the protein-protein interfaces by a variety of techniques. Our work studying the mechanism of chaperone function will inform the design and interpretation of results as we probe the importance of each oligomerization interface.  Raysa Cabrejo '14 studied the stoichiometry of the interactions between ABC and insulin.  Yusrah Kaudeer '21 plans to do further studies of the 3E mutant using both light scattering and microscopy, Kashmeera Baboolall '20 thesis explored the assembly and subunit exchange using both bulk and single molecule FRET.  Alex Dreisbach '17 studied the effects of chloride ion and choline on the aggregation behavior as studied by single molecule spectroscopyl  Alex Pearlman '13 focused on the interactions of the palindromic section of the C-terminal strands.  Using small peptides that mimicked the C terminal strands labelled with donor dyes, he studied their assembly with a mutant ABC with a cysteine residue inserted into the C terminal strand of an intact protein which he labelled with acceptor dye. Jean Santos '12 did some early studies examining differences in mixed oligomerization states of ABC and AAC as determined by the insulin scattering assay.

Yusrah Kaudeer '21

Alpha Crystallin story got covered in The Student but even before that we knew she was a superstar. 

Kashmeera Baboolall '20 Thesis

Despite the setbacks of COVID-19, Kashew put together an amazing thesis combining the results of her bulk light scattering work, her single molecule FRET studies in our lab (in which she re-assembled to single molecule spectrometer taken apart for our move to the new science building) and in which she collaborated with Dr. Anne Gershenson at UMass to do FCCS work. 

Thesis Title: Probing the pH Modulation of a Hyper-Phosphorylated Model of Alpha-B Crystallin


Biophysical Society Meeting Poster -2020

Enhanced pH Dependent Modulation of Alpha Crystallin Chaperone Function And Subunit Exchange In An N-Terminal Phosphorylation Mimic

Kashmeera Baboolall,Yusrah Kaudeer, Anne Gershenson, Patricia B. O'Hara, Biophysical Society Meeting February 2020, Biophysical Journal 118(3):510a; pH Dependence of Oligomerization And Functional Activity Of Alpha B Crystallin DOI: 10.1016/j.bpj.2019.11.2809

The functional behavior of the small heat shock protein, Alpha B Crystallin, can be modulated through the acquisition of different oligomerization states as a function of many environmental factors.   One example is stress induced phosphorylation, which can affect the assembly of the monomer into higher order structures.   A 3E mutant (S15E, S45E, and S59E) is used as a proxy for the phosphorylated wild type system.  Chaperone activity of both wild type and 3E mutants can be measured as a function of pH.  Here the inhibition of insulin aggregation in the presence of the ABC is treated as a model system for chaperone function in vivo. After addition of dithiothreitol, the insulin aggregates and scatter light.  Looking at both wild type and mutant ABC showed marked pH dependence with optimum function observed at pH 7.4. 

Fluorescence Correlation Spectroscopy (FCS) can be used to measure the size and size distribution of both the wild type and 3E mutant as a function of pH.  Here we see

Biophysical Society Meeting Poster -2019


Kashmeera Baboolall, Belelot Birhanu, Natalie Braun, Yusrah Kaudeer, Patricia B. O'Hara, Biophysical Society Meeting February 2019, Biophysical Journal 116(3):190a DOI:10.1016/j.bpj.2018.11.1051 Enhanced pH Dependent Modulation of Alpha Crystallin Chaperone Function And Subunit Exchange In An N-Terminal Phosphorylation Mimic

 It has been hypothesized that N-terminal phosphorylation can modulate chaperone function of alpha crystallin in vitro. We used an alpha crystallin mutant in which 3 serines at positions 19, 45 and 59 have been replaced by glutamates. Since the negative charges of the 3 glutamate residues at neutral pH mimic the phosphorylation that can happen in vivo, the mutant is referred to as the 3E phosphorylation mimic. pH can play a role in the modulation of chaperone function of the phosphorylated protein the 3E phosphorylation mimic. We measured the capacity of wild type and mutant protein to modulate insulin aggregation at pH 5 (acidic), pH 7 (neutral), and pH 8 (basic). Light scattering produced upon insulin denaturation can be followed as a function of time to determine the rate of denaturation and extent of its disruption by both wild type and the 3E phosphorylation mimic. We see that aggregation is dependent on pH for both wild type and the 3E phosphorylation mimic, with chaperone function being lowest under acidic conditions and rising at neutral and basic conditions. Above pH 7, chaperone behavior of the 3E phosphorylation mimic far exceeds wild type. We have also attached fluorescent labels to both wild type and 3E mutant in order to explore the exchange of the alpha crystallin subunits within the protein multimer. By observing changes in FRET when one solution labelled with FRET-donor is mixed with a solution labelled with FRET-acceptor, we can measure the extent of the subunit exchange and determine how exchange depends upon pH for both wild type and the 3E phosphorylation mimic. We can infer that in vivo phosphorylation of alpha crystallin can be used to modulate chaperone function in stressed cells.

Interaction of α-Crystallin B in Functional Oligomerization - R.Cabrejo '14

Biophysical Journal, 2014, Vol.106 (2) DOI 10.1016/j.bpj.2013.11.2666

Alpha-Crystallin-B (aXB) is a small heat shock protein found mainly in the lens of the eye. There it serves two purposes: acting as a chaperone to prevent the misfolding of the other protein and contributing to the high protein concentrations required to focus light. When aXB chaperone function fails cataract formation begins. Understanding the dependence of the mechanism of aXB chaperone function on its oligomerization state will aid in the delay or prevention of cataracts. Oligomerization of alpha-crystallinB is pH dependent. We present a mechanism for the chaperone function of aXB using insulin as a model system in which it is possible to induce aggregation. The relationship between oligomerization and chaperone function is tested by measuring the dependence of the onset of light scattering insulin aggregates on the oligomerization state of the aXB. Different oligomerization states can be induced by pre-incubating aXB at a range of pHs. Attenuated chaperone function is observed for aXB pre-incubated at elevated pH even when the assay itself takes place at pH 7. These results are consistent with a model in which aXB is kinetically trapped into higher oligomer states at high pH and unable to return quickly to its functional equilibrium state upon dilution to neutral pH. We will also test the generality of this model by extending our studies to Gamma-Crystallin. Finally, we will measure the contribution of the C-terminal strand to chaperone function and oligomerization using fluorescence resonance energy transfer (FRET). Observation of pH dependent dynamics of the C-terminal strand will help to distinguish between strand-exchanged and dynamic states of the C-terminus, allowing its contribution to oligomerization and chaperone function to be probed.

Characterizing Palindromic Strand Exchange in α-Crystallin Oligomerization - A. Pearlman '13

Biophysical Journal, 2013, Vol. 104, p394a;  DOI:

Alpha-crystallin, a member of the small heat shock protein (sHSP) class, is expressed in eye lens, muscle, and brain tissue where it contributes to homeostasis by chaperoning and thus preventing the pathogenic aggregation of damaged or unfolded proteins. As a major cytoplasmic component in the eye lens, alpha-crystallin is further essential for producing a high index of refraction. Despite its high concentrations, alpha-crystallin does not itself aggregate or crystallize, a property facilitated by its populating dynamic, polydisperse assemblies. Oligomerization in this manner is a common feature of the sHSPs and is likely connected to their chaperone function.

Structural studies1 suggest that one mechanism of polydispersity may be the ability of a palindromic sequence centered on the sHSP IXI motif in the alphaB-crystallin (αB) isoform c-terminus to bind bidirectionally to other monomers. Bidirectional strand exchanges would result in heterogeneous oligomeric structures while maintaining near identical residue interactions.

Modeling Disfunction in α-Crystallin-J.Santos '12


Alpha Crystallin is the major protein component of the human lens and plays an important role in the prevention of cataracts. α -Crystallin (α X) oligomers consist of two isoforms, α X-A and α X-B which share high sequence similarity and define the common α -Crystallin fold found in many small heat shock proteins (sHSPs). α X-A and α X-B are hypothesized to play two important roles within the lens. First, α X-A and α X-B belong to a group of proteins called Crystallins (α , β , and γ ) that are very stable proteins that play a role in preserving a uniform density within the lens, which allows it to focus light. The Crystallin proteins’ ability to form diverse and stable oligomers results in a glass-like rather than crystalline organization to the lens protein material, which also aids in the long-term stability of this high-density protein organ. Second, α X-A and α X-B both function as sHSPs that bind to misfolded proteins, preventing formation of large, insoluble protein aggregates (the beginning of cataracts). Our lab is investigating the molecular interactions between α X-A and α X-B that result in its stability, diverse oligomerization, and chaperone function. To this end we are using a model, inducible misfolding protein (insulin B-chain) to study chaperone function by light scatter under various conditions. We are also using random and targeted modification of α X-A and α X-B to simulate long-term protein damage and degradation observed in aged lenses. Focus on the C-terminal strand exchange observed in recent crystal structures and proposed to aid in α X-A and α X-B polydisperse oligomerization is additionally aiding experimental design. We hope to identify specific molecular interactions that result in α X-A and α X-B’s chaperone function, and determine how those interactions relate to stability and self-oligomerization.