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α-crystallin is a major protein found in the mammalian eye lens that works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress in the eye lens. These functions of α-crystallin are significant for maintaining lens transparency. However, with age and cataract formation, the concentration of α-crystallin in the eye lens cytoplasm decreases with a corresponding increase in the membrane-bound α-crystallin, accompanied by increased light scattering. The purpose of this review is to summarize previous and recent findings of the role of the: (1) lens membrane components, i.e., the major phospholipids (PLs) and sphingolipids, cholesterol (Chol), cholesterol bilayer domains (CBDs), and the integral membrane proteins aquaporin-0 (AQP0; formally MIP26) and connexins, and (2) α-crystallin mutations and post-translational modifications (PTMs) in the association of α-crystallin to the eye lens’s fiber cell plasma membrane, providing thorough insights into a molecular basis of such an association. Furthermore, this review highlights the current knowledge and need for further studies to understand the fundamental molecular processes involved in the association of α-crystallin to the lens membrane, potentially leading to new avenues for preventing cataract formation and progression.

α-Crystallin (αABc) is a major protein comprised of αA-crystallin (αAc) and αB-crystallin (αBc) that is found in the human eye lens and works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress. However, with age and cataract formation, the concentration of αABc in the eye lens cytoplasm decreases, with a corresponding increase in the membrane-bound αABc. This study uses the electron paramagnetic resonance (EPR) spin-labeling method to investigate the role of cholesterol (Chol) and Chol bilayer domains (CBDs) in the binding of αAc, αBc, and αABc to the Chol/model of human lens-lipid (Chol/MHLL) membranes. The maximum percentage of membrane surface occupied (MMSO) by αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trends: MMSO (αAc) > MMSO (αBc) ≈ MMSO (αABc), indicating that a higher amount of αAc binds to these membranes compared to αBc and αABc. However, with an increase in the Chol concentration in the Chol/MHLL membranes, the MMSO by αAc, αBc, and αABc decreases until it is completely diminished at a mixing ratio of 1.5. The Ka of αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trend: Ka (αBc) ≈ Ka (αABc) > Ka (αAc), but it was close to zero with the diminished binding at a Chol/MHLL mixing ratio of 1.5. The mobility near the membrane headgroup regions decreased with αAc, αBc, and αABc binding, and the Chol antagonized the capacity of the αAc, αBc, and αABc to decrease mobility near the headgroup regions. No significant change in membrane order near the headgroup regions was observed, with an increase in αAc, αBc, and αABc concentrations. Our results show that αAc, αBc, and αABc bind differently with Chol/MHLL membranes at mixing ratios of 0 and 0.5, decreasing the mobility and increasing hydrophobicity near the membrane headgroup region, likely forming the hydrophobic barrier for the passage of polar and ionic molecules, including antioxidants (glutathione), creating an oxidative environment inside the lens, leading to the development of cataracts. However, all binding was completely diminished at a mixing ratio of 1.5, indicating that high Chol and CBDs inhibit the binding of αAc, αBc, and αABc to membranes, preventing the formation of hydrophobic barriers and likely protecting against cataract formation.

Eye lens α-crystallin has been shown to become increasingly membrane-bound with age and cataract formation; however, to our knowledge, no studies have investigated the membrane interactions of α-crystallin throughout the development of cataracts in separated cortical membrane (CM) and nuclear membrane (NM) from single human lenses. In this study, four pairs of human lenses from age-matched male and female donors and one pair of male lenses ranging in age from 64 to 73 years old (yo) were obtained to investigate the interactions of α-crystallin with the NM and CM throughout the progression of cortical cataract (CC) and nuclear cataract (NC) using the electron paramagnetic resonance spin-labeling method. Donor health history information (diabetes, smoker, hypertension, radiation treatment), sex, and race were included in the data analysis. The right eye lenses CM and NM investigated were 64 yo male (CC: 0), 68 yo male (CC: 3, NC: 2), 73 yo male (CC: 1, NC: 2), 68 yo female (CC: 3, NC: 2), and 73 yo female (CC: 1, NC: 3). Similarly, left eye lenses CM and NM investigated were 64 yo male (CC: 0), 68 yo male (CC: 3, NC: 2), 73 yo male (CC: 2, NC: 3), 68 yo female (CC: 3, NC: 2), and 73 yo female (CC: 1, NC: 3). Analysis of α-crystallin binding to male and female eye lens CM and NM revealed that the percentage of membrane surface occupied (MSO) by α-crystallin increases with increasing grade of CC and NC. The binding of α-crystallin resulted in decreased mobility, increased order, and increased hydrophobicity on the membrane surface in male and female eye lens CM and NM. CM mobility decreased with an increase in cataracts for both males and females, whereas the male lens NM mobility showed no significant change, while female lens NM showed increased mobility with an increase in cataract grade. Our data shows that a 68 yo female donor (long-term smoker, pre-diabetic, and hypertension; grade 3 CC) showed the largest MSO by α-crystallin in CM from both the left and right lens and had the most pronounced mobility changes relative to all other analyzed samples. The variation in cholesterol (Chol) content, size and amount of cholesterol bilayer domains (CBDs), and lipid composition in the CM and NM with age and cataract might result in a variation of membrane surface mobility, membrane surface hydrophobicity, and the interactions of α-crystallin at the surface of each CM and NM. These findings provide insight into the effect of decreased Chol content and the reduced size and amount of CBDs in the cataractous CM and NM with an increased binding of α-crystallin with increased CC and NC grade, which suggests that Chol and CBDs might be a key component in maintaining lens transparency.

Several discoveries show that with age and cataract formation, β-crystallin binds with the lens membrane or associates with other lens proteins, which bind with the fiber cell plasma membrane, accompanied by light scattering and cataract formation. However, how lipids (phospholipids and sphingolipids) and cholesterol (Chol) influence β-crystallin binding to the membrane is unclear. This research aims to elucidate the role of lipids and Chol in the binding of β-crystallin to the membrane and the membrane’s physical properties (mobility, order, and hydrophobicity) with β-crystallin binding. We used electron paramagnetic resonance (EPR) spin-labeling methods to investigate the binding of βL-crystallin with a model of porcine lens-lipid (MPLL), model of mouse lens-lipid (MMLL), and model of human lens-lipid (MHLL) membrane with and without Chol. Our results show that βL-crystallin binds with all of the investigated membranes in a saturation manner, and the maximum parentage of the membrane surface occupied (MMSO) by βL-crystallin and the binding affinity (Ka) of βL-crystallin to the membranes followed trends: MMSO (MPLL) > MMSO (MMLL) > MMSO (MHLL) and Ka (MHLL) > Ka (MMLL) ≈ Ka (MPLL), respectively, in which the presence of Chol reduces the MMSO and Ka for all membranes. The mobility near the headgroup regions of the membranes decreases with an increase in the binding of βL-crystallin; however, the decrease is more pronounced in the MPLL and MMLL membranes than the MHLL membrane. In the MPLL and MMLL membranes, the membranes become slightly ordered near the headgroup with an increase in βL-crystallin binding compared to the MHLL membrane. The hydrophobicity near the headgroup region of the membrane increases with βL-crystallin binding; however, the increase is more pronounced in the MPLL and MMLL membranes than the MHLL membrane, indicating that βL-crystallin binding creates a hydrophobic barrier for the passage of polar molecules, which supports the barrier hypothesis in cataract formation. However, in the presence of Chol, there is no significant increase in hydrophobicity with βL-crystallin binding, suggesting that Chol prevents the formation of a hydrophobic barrier, possibly protecting against cataract formation.

α-crystallin-membrane association increases with age and cataracts, with the primary association site of α-crystallin being phospholipids. However, it is unclear if phospholipids’ acyl chain length and degree of unsaturation influence α-crystallin association. We used the electron paramagnetic resonance approach to investigate the association of α-crystallin with phosphatidylcholine (PC) membranes of different acyl chain lengths and degrees of unsaturation and with and without cholesterol (Chol). The association constant (Ka) of α-crystallin follows the trends, i.e., Ka (14:0–14:0 PC) > Ka (18:0–18:1 PC) > Ka (18:1–18:1 PC) ≈ Ka (16:0–20:4 PC) where the presence of Chol decreases Ka for all membranes. With an increase in α-crystallin concentration, the saturated and monounsaturated membranes rapidly become more immobilized near the headgroup regions than the polyunsaturated membranes. Our results directly correlate the mobility and order near the headgroup regions of the membrane with the Ka, with the less mobile and more ordered membrane having substantially higher Ka. Furthermore, our results show that the hydrophobicity near the headgroup regions of the membrane increases with the α-crystallin association, indicating that the α-crystallin-membrane association forms the hydrophobic barrier to the transport of polar and ionic molecules, supporting the barrier hypothesis in cataract development.

The lens of the eye loses elasticity with age, while α-crystallin association with the lens membrane increases with age. It is unclear whether there is any correlation between α-crystallin association with the lens membrane and loss in lens elasticity. This research investigated α-crystallin membrane association using atomic force microscopy (AFM) for the first time to study topographical images and mechanical properties (breakthrough force and membrane area compressibility modulus (KA), as measures of elasticity) of the membrane. α-Crystallin extracted from the bovine lens cortex was incubated with a supported lipid membrane (SLM) prepared on a flat mica surface. The AFM images showed the time-dependent interaction of α-crystallin with the SLM. Force spectroscopy revealed the presence of breakthrough events in the force curves obtained in the membrane regions where no α-crystallin was associated, which suggests that the membrane’s elasticity was maintained. The force curves in the α-crystallin submerged region and the close vicinity of the α-crystallin associated region in the membrane showed no breakthrough event within the defined peak force threshold, indicating loss of membrane elasticity. Our results showed that the association of α-crystallin with the membrane deteriorates membrane elasticity, providing new insights into understanding the molecular basis of lens hardening and presbyopia.

Several studies reported that α-crystallin concentrations in the eye lens cytoplasm decrease with a corresponding increase in membrane-bound α-crystallin with age and cataracts. The influence of the lipid and cholesterol composition difference between cortical membrane (CM) and nuclear membrane (NM) on α-crystallin binding to membranes is still unclear. This study uses the electron paramagnetic resonance (EPR) spin-labeling method to investigate the α-crystallin binding to bovine CM and NM derived from the total lipids extracted from a single lens. Compared to CMs, NMs have a higher percentage of membrane surface occupied by α-crystallin and binding affinity, correlating with less mobility and more order below and on the surface of NMs. α-Crystallin binding to CM and NM decreases mobility with no significant change in order and hydrophobicity below and on the surface of membranes. Our results suggest that α-crystallin mainly binds on the surface of bovine CM and NM and such surface binding of α-crystallin to membranes in clear and young lenses may play a beneficial role in membrane stability. However, with decreased cholesterol content within the CM, which mimics the decreased cholesterol content in the cataractous lens membrane, α-crystallin binding increases the hydrophobicity below the membrane surface, indicating that α-crystallin binding forms a hydrophobic barrier for the passage of polar molecules, supporting the barrier hypothesis in developing cataracts.

Several discoveries show that with age and cataract formation, β-crystallin binds with the lens membrane or associates with other lens proteins, which bind with the fiber cell plasma membrane, accompanied by light scattering and cataract formation. However, how lipids (phospholipids and sphingolipids) and cholesterol (Chol) influence β-crystallin binding to the membrane is unclear. This research aims to elucidate the role of lipids and Chol in the binding of β-crystallin to the membrane and the membrane’s physical properties (mobility, order, and hydrophobicity) with β-crystallin binding. We used electron paramagnetic resonance (EPR) spin-labeling methods to investigate the binding of β[sub.L]-crystallin with a model of porcine lens-lipid (MPLL), model of mouse lens-lipid (MMLL), and model of human lens-lipid (MHLL) membrane with and without Chol. Our results show that β[sub.L]-crystallin binds with all of the investigated membranes in a saturation manner, and the maximum parentage of the membrane surface occupied (MMSO) by β[sub.L]-crystallin and the binding affinity (K[sub.a]) of β[sub.L]-crystallin to the membranes followed trends: MMSO (MPLL) > MMSO (MMLL) > MMSO (MHLL) and K[sub.a] (MHLL) > K[sub.a] (MMLL) ≈ K[sub.a] (MPLL), respectively, in which the presence of Chol reduces the MMSO and K[sub.a] for all membranes. The mobility near the headgroup regions of the membranes decreases with an increase in the binding of β[sub.L]-crystallin; however, the decrease is more pronounced in the MPLL and MMLL membranes than the MHLL membrane. In the MPLL and MMLL membranes, the membranes become slightly ordered near the headgroup with an increase in β[sub.L]-crystallin binding compared to the MHLL membrane. The hydrophobicity near the headgroup region of the membrane increases with β[sub.L]-crystallin binding; however, the increase is more pronounced in the MPLL and MMLL membranes than the MHLL membrane, indicating that β[sub.L]-crystallin binding creates a hydrophobic barrier for the passage of polar molecules, which supports the barrier hypothesis in cataract formation. However, in the presence of Chol, there is no significant increase in hydrophobicity with β[sub.L]-crystallin binding, suggesting that Chol prevents the formation of a hydrophobic barrier, possibly protecting against cataract formation.

Electrostatic interactions are significant for biomolecules given the prevalence of electric charges in all major families of biomolecules (e.g., DNA/RNA, proteins, and lipids). Precise and predictable knowledge of biomolecular electrostatics has thus been a longstanding goal in biophysics. While the physical laws of electrostatics are well-established, biomolecular electrostatics presents formidable challenges due to the sheer size of the multi-component system in a salty aqueous environment. As a result, our understanding of biomolecular electrostatics is far from complete, despite extensive efforts in the past decades. My dissertation research focuses on the model system of nucleic acids helices and aims to elucidate the role of molecular structure, largely ignored up to now, in electrostatic interactions. Specifically, we probe how structures of nucleic acids and peptides affect the electrostatic interactions between nucleic acid helices by osmotic stress method and x-ray diffraction (XRD). Different DNA/RNA sequences and surface modifications are used to produce the various helical structures of nucleic acids, osmotic stress is used to vary the forces between nucleic acid helices (DNA-DNA or RNA-RNA), and XRD is used to measure the inter-helical spacing. Consequently, our measurement yields the quantitative relationship between DNA-DNA force and DNA-DNA spacing. Our first-of-its-kind measurement discovers and quantifies structure-dependent behaviors in force-spacing relations of DNA-DNA and RNA-RNA interactions that appear to be strongly correlated with the incurred structural changes. Furthermore, DNA-DNA forces modulated by the peptides, selected from histone tails with different charges and sequences are measured. The force curves are found to depend on both the total charge and sequence of the peptides, revealing specific (peptide-sequence modulated) and non-specific (charge modulated) interactions with DNA. More broadly, our observations bear biological implications concerning chromatin conformation and epigenetic regulations, and possible future directions include studies of additional biological nucleic acids and peptides sequences, molecular dynamics simulations (MD), and bioinformatic analyses.

Probing the role of surface structure in electrostatic interactions, we report the first observation of sequence-dependent dsDNA condensation by divalent alkali cations. Disparate behaviors were found between two repeating sequences with 100% AT content, a poly(A)-poly(T) duplex (AA-TT) and a poly(AT)-poly(TA) duplex (AT-TA). While AT-TA exhibits non-distinguishable behaviors from random-sequence genomic DNA, AA-TT condenses in all divalent alkali ions (Mg2+, Ca2+, Sr2+, and Ba2+). We characterized these interactions experimentally and investigated the underlying principles using all-atom computer simulations. Both experiment and simulation demonstrate that AA-TT condensation is driven by nonspecific ion-DNA interactions, which depend on the structures of ions and DNA surface. Detailed analyses reveal sequence-enhanced major groove binding (SEGB) of point-charged alkali ions as the major difference between AA-TT and AT-TA, which originates from the continuous and close stacking of nucleobase partial charges in AA-TT but not in AT-TA. These SEGB cations elicit attraction via spatial correlations with the phosphate backbone of neighboring helices, reminiscent of the DNA-zipper model, which though assumes a non-electrostatic cation groove binding a priori. Our study thus presents a distinct molecular mechanism of DNA-DNA interaction in which sequence-directed surface motifs act with abundant divalent alkali cations non-specifically to enact sequence-dependent behaviors. This physical insight allows a renewed understanding of the function of repeating DNA sequences in genome organization and regulation and offers a facile approach for DNA technology to control the assembly process of DNA nanostructures.