Keith M Berland

Emory University, Atlanta, Georgia, United States

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Publications (55)137.86 Total impact

  • [Show abstract] [Hide abstract] ABSTRACT: Living cells contain a range of densely phosphorylated surfaces, including phospholipid membranes, ribonucleoproteins, and nucleic acid polymers. Hyperphosphorylated surfaces also accumulate in neurodegenerative diseases as neurofibrillar tangles. We have synthesized and structurally characterized a precisely patterned phosphotyrosine surface and establish this assembly as a surrogate of the neuronal tangles by demonstrating its high-affinity binding to histone H1. This association with nucleic acid binding proteins underscores the role such hyperphosphorylated surfaces may play in disease and opens functional exploration into protein-phosphorylated surface interactions in a wide range of other complex assemblies.
    No preview · Article · Jun 2014 · Biochemistry
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    Neil R Anthony · Anil K Mehta · David G Lynn · Keith M Berland
    [Show abstract] [Hide abstract] ABSTRACT: The cross-β peptide architecture is associated with numerous functional biomaterials and deleterious disease related aggregates. While these diverse and ubiquitous paracrystalline assemblies have been widely studied, a fundamental understanding of the nucleation and aggregation pathways to these structures remains elusive. Here we highlight a novel application of fluorescence lifetime imaging microscopy in characterising the critical stages of peptide aggregation. Using the central nucleating core of the amyloid-β (Aβ), Aβ(16-22), as a model cross-β system, and utilising a small fraction of rhodamine labelled peptide (Rh110-Aβ(17-22)), we map out a folding pathway from monomer to paracrystalline nanotube. Using this intrinsic fluorescence reporter, we demonstrate the effects of interfaces and evaporation on the nucleation of sub-critical concentration solutions, providing access to previously uncharacterised intermediate morphologies. Using fluorescence lifetime we follow the local peptide environment through the stages of nucleation and hydrophobic collapse, ending in a stable final structure. This work provides a metric for future implementations of measuring fluorescence lifetimes of intrinsic fluorescence reporters during the very dynamic processes relating to peptide nucleation and maturation.
    Full-text · Article · Apr 2014 · Soft Matter
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    Neil R Anthony · Keith M Berland
    [Show abstract] [Hide abstract] ABSTRACT: Fluorescence fluctuation methods have become invaluable research tools for characterizing the molecular-level physical and chemical properties of complex systems, such as molecular concentrations, dynamics, and the stoichiometry of molecular interactions. However, information recovery via curve fitting analysis of fluctuation data is complicated by limited resolution and challenges associated with identifying accurate fit models. We introduce a new approach to fluorescence fluctuation spectroscopy that couples multi-modal fluorescence measurements with multi-modal global curve fitting analysis. This approach yields dramatically enhanced resolution and fitting model discrimination capabilities in fluctuation measurements. The resolution enhancement allows the concentration of a secondary species to be accurately measured even when it constitutes only a few percent of the molecules within a sample mixture, an important new capability that will allow accurate measurements of molecular concentrations and interaction stoichiometry of minor sample species that can be functionally important but difficult to measure experimentally. We demonstrate this capability using τFCS, a new fluctuation method which uses simultaneous global analysis of fluorescence correlation spectroscopy and fluorescence lifetime data, and show that τFCS can accurately recover the concentrations, diffusion coefficients, lifetimes, and molecular brightness values for a two component mixture over a wide range of relative concentrations.
    Full-text · Article · Feb 2014 · PLoS ONE
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    Neil Anthony · Keith Berland
    [Show abstract] [Hide abstract] ABSTRACT: Fluorescence correlation spectroscopy (FCS) and related fluctuation spectroscopy and microscopy methods have become important research tools that enable detailed investigations of the chemical and physical properties of molecules and molecular systems in a variety of complex environments. Information recovery via curve fitting of fluctuation data can present complicating challenges due to limited resolution and/or problems with fitting model verification. We discuss a new approach to data analysis called τFCS that couples multiple modes of signal acquisition, here specifically FCS and fluorescence lifetimes, with global analysis. We demonstrate enhanced resolution using τFCS, including the capability to recover the concentration of both molecular species in a two-component mixture even when the species have identical diffusion coefficients and molecular brightness values, provided their fluorescent lifetimes are distinct. We also demonstrate how τFCS provides useful tools for model discrimination in FCS curve fitting.
    Full-text · Article · Dec 2013 · Methods in enzymology
  • [Show abstract] [Hide abstract] ABSTRACT: Abstract Advances in electron and fluorescence microscopy imaging are now providing molecular-level resolution of macromolecular and supramolecular dynamic self-organization. Nowhere has this information been more impactful than in understanding the increasing diversity of biomolecular misfolding diseases. The amyloid-β protein associated with Alzheimer’s disease and its congeners have now offered insight into both neurodegenerative disease as well as functional scaffolds for constructing bio-inspired nanomaterials. Here we discuss the exploitation of these methods within these dynamic networks of paracrystalline supramolecular phases, and define minimal nucleation events, resolve the templated propagation events, and reveal the complex dynamic molecular networks of these polymorphic assemblies.
    No preview · Chapter · Nov 2013
  • Keith M. Berland · Neil R. Anthony
    [Show abstract] [Hide abstract] ABSTRACT: Fluorescence correlation spectroscopy (FCS) and related fluctuation spectroscopy and microscopy methods have become important research tools that enable detailed investigations of the chemical and physical properties of molecules and molecular systems in a variety of complex environments. When analyzed successfully fluctuation measurements often provide unique information that is otherwise difficult to measure, such as molecular concentrations and interaction stoichiometry. However, information recovery via curve fitting of fluctuation data can present challenges due to limited resolution and/or problems with fitting model verification. We discuss a new approach to fluctuation data analysis coupling multi-modal fluorescence measurements and global analysis, and demonstrate how this approach can provide enhanced sensitivity and resolution in fluctuation measurements. We illustrate the approach using a combination of FCS and fluorescence lifetime measurements, here called τFCS, and demonstrate the capability to recover the concentration of two independent molecular species in a two component mixture even when the species have identical diffusion coefficients and molecular brightness values. This work was partially supported by NSF grants MCB0817966 and DMR0907435.
    No preview · Article · Feb 2013 · Proceedings of SPIE - The International Society for Optical Engineering
  • Jed Brody · Daniel Weiss · Keith Berland
    [Show abstract] [Hide abstract] ABSTRACT: We introduce a visually appealing experimental demonstration of Fresnel reflection. In this simple optical experiment, a polarized light beam travels through a high numerical-aperture microscope objective, reflects off a glass slide, and travels back through the same objective lens. The return beam is sampled with a polarizing beam splitter and produces a surprising geometric pattern on an observation screen. Understanding the origin of this pattern requires careful attention to geometry and an understanding of the Fresnel coefficients for S and P polarized light. We demonstrate that in addition to a relatively simple experimental implementation, the shape of the observed pattern can be computed both analytically and by using optical modeling software. The experience of working through complex mathematical computations and demonstrating their agreement with a surprising experimental observation makes this a highly educational experiment for undergraduate optics or advanced-lab courses. It also provides a straightforward yet non-trivial system for teaching students how to use optical modeling software.
    No preview · Article · Jan 2013 · American Journal of Physics
  • [Show abstract] [Hide abstract] ABSTRACT: Recent evidence suggests that simple peptides can access diverse amphiphilic phases, and that these structures underlie the robust and widely distributed assemblies implicated in nearly 40 protein misfolding diseases. Here we exploit a minimal nucleating core of the Aβ peptide of Alzheimer's disease to map its morphologically accessible phases that include stable intermolecular molten particles, fibers, twisted and helical ribbons, and nanotubes. Analyses with both fluorescence lifetime imaging microscopy (FLIM) and transmission electron microscopy provide evidence for liquid-liquid phase separations, similar to the coexisting dilute and dense protein-rich liquid phases so critical for the liquid-solid transition in protein crystallization. We show that the observed particles are critical for transitions to the more ordered cross-β peptide phases, which are prevalent in all amyloid assemblies, and identify specific conditions that arrest assembly at the phase boundaries. We have identified a size dependence of the particles in order to transition to the para-crystalline phase and a width of the cross-β assemblies that defines the transition between twisted fibers and helically coiled ribbons. These experimental results reveal an interconnected network of increasing molecularly ordered cross-β transitions, greatly extending the initial computational models for cross-β assemblies.
    No preview · Article · Mar 2012 · Langmuir
  • [Show abstract] [Hide abstract] ABSTRACT: We use Fluorescence Lifetime Imaging Microscopy (FLIM) and Second Harmonic Imaging Microscopy (SHIM) to investigate the fundamental molecular mechanisms responsible for nucleation and growth of amyloidogenic-derived nanomaterials. The nanomaterials are assembled from of Amyloid-β(16-22), specifically Ac-KLVFFAE-NH2, the nucleating core of the Alzheimer's Amyloid-β protein. We describe how FLIM and SHIM can be used to follow different nucleation pathways and to quantify structural heterogeneities within these complex nanomaterials. New evidence suggests that different structures emerge from distinct nucleation pathways and these insights inform our understanding of the peptide self-assembly mechanisms. We discuss these insights in the context of a top down understanding of amyloidogenic diseases, the bottom up control of functional nanomaterials and the discovery of realtime structural indicators for nanofabrication strategies.
    No preview · Article · Feb 2012 · Proceedings of SPIE - The International Society for Optical Engineering
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    Neil R. Anthony · David G. Lynn · Keith M. Berland
    Full-text · Article · Jan 2012 · Biophysical Journal
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    Neil R. Anthony · David G. Lynn · Keith Berland
    Full-text · Article · Feb 2011 · Biophysical Journal
  • P. T. C. So · K. Duggar · K. M. Berland · E. Gratton
    [Show abstract] [Hide abstract] ABSTRACT: A new technique for determining cellular viscosity has been developed based on two-photon excitation and fluorescence correlation spectroscopy. We have applied this method to map intracellular diffusion coefficient of small molecules at selected cytoplasmic locations of mouse fibroblast cells. The cytoplasmic diffusion rate is one tenth of that in an aqueous environment.
    No preview · Article · Jan 2011 · MRS Online Proceeding Library
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    Yan Liang · David G Lynn · Keith M Berland
    [Show abstract] [Hide abstract] ABSTRACT: Access to native protein structure depends on precise polypeptide folding and assembly pathways. Identifying folding missteps that may lead to the nearly 40 protein misfolding diseases could feature prominently in the development of intervention strategies. Accordingly, we have investigated the earliest steps of assembly by the folding nucleus of the Alzheimer's disease Abeta peptide with real-time imaging and fluorescence correlation spectroscopy. These analyses reveal the immediate formation of large micrometer size clusters maintaining properties of intermolecular molten globules. These dynamic unstructured aggregates serve as the nucleating sites for amyloid growth and, as with native protein folding, appear important for backbone desolvation. The resulting amyloid nucleus however is able to template monomer addition from solution at rates from 2K peptides/s at millimolar peptide concentrations. This direct observation of amyloid assembly unifies several divergent models that currently exist for protein misfolding.
    Full-text · Article · May 2010 · Journal of the American Chemical Society
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    Keith Berland · Yan Liang · Neil Anthony
    Preview · Article · Jan 2010 · Biophysical Journal
  • Neil Anthony · Keith Berland
    [Show abstract] [Hide abstract] ABSTRACT: Models used in fluorescence correlation spectroscopy (FCS) generally assume diffusion of a standard Fickian nature such that the mean square displacement (MSD,) of the molecular diffusion is linearly proportional to time, i.e. t. In complex systems the diffusion can be anomalous, which is commonly described via a power law dependence of the MSD, i.e. t^alpha. When measuring anomalous dynamics using FCS, the correlation functions are typically measured over a single lengthscale and the anomalous exponent, alpha, is recovered through curve fitting. The anomalous exponent accurately describes the time dependence of the diffusion over the measurement lengthscale, yet for the majority of experimental systems it has not yet been tested whether the dynamics predicted by the fit are actually observed over different lengthscales -- i.e. whether or not the assumed power law dynamics truly describe the system dynamics. We investigate using scanning FCS methods that simultaneously measure correlation functions over a range of lengthscales in order to determine how accurately the physical models describe the dynamics. We use simulations to test these methods and discuss their application for measuring drug delivery rates in biomedical hydrogels.
    No preview · Article · Nov 2009
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    Peng Guo · Keith M. Berland
    [Show abstract] [Hide abstract] ABSTRACT: Fluorescence correlation spectroscopy (FCS) has been widely used investigate molecular dynamics and interactions in biological systems. FCS typically resolves the component species of a sample either through differences in diffusion coefficient or molecular brightness. Diffusion based assays currently have a major limitation which requires that the diffusion coefficients of component species in a sample must be substantially different in order to be resolved. This criterion is not met in many important cases, such as when molecules of similar molecular weight bind to each other. This limitation can be overcome, and resolution of FCS measurements enhanced, by combining FCS measurements with measurements of fluorescence lifetimes. By using of global analysis on simultaneously acquired FCS and lifetime data we show that we can dramatically enhance resolution in FCS measurements, and accurately resolve the concentration and diffusion coefficients of multiple sample components even when their diffusion coefficients are identical provided there is a difference in the lifetime of the component species. We show examples of this technique using both simulations and experiments. It is expected that this method will be of significance for binding assays studying molecular interactions.
    Preview · Article · Nov 2009 · Biophysical Journal
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    Keith Berland · David G. Lynn · Yan Liang
    [Show abstract] [Hide abstract] ABSTRACT: Nucleation and growth mechanisms in amyloid materials are resolved using single molecule fluorescence imaging and spectroscopy. Results identify an intermolecular molten globule state as a key intermediate of the nucleation pathway.
    Full-text · Article · Oct 2009
  • Neil Anthony · Keith Berland · Peng Guo
    No preview · Chapter · Jul 2009
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    Jianrong Wu · Anita H Corbett · Keith M Berland
    [Show abstract] [Hide abstract] ABSTRACT: We have investigated classical nuclear localization sequence (NLS) mediated protein trafficking by measuring biomolecular dynamics within living cells using two-photon fluorescence correlation spectroscopy. By directly observing the behavior of specific molecules in their native cellular environment, it is possible to uncover functional details that are not apparent from traditional biochemical investigations or functional assays. We show that the intracellular mobility of NLS cargoes and their import receptor proteins, karyopherin-alpha and karyopherin-beta, can be robustly measured and that quantitative comparison of intracellular diffusion coefficients provides new insights into nuclear transport mechanisms. Import cargo complexes are assembled throughout the cytoplasm, and their diffusion is slower than predicted by molecular weight due to specific interactions. Analysis of NLS cargo diffusion in the cytoplasm indicates that these interactions are likely disrupted by NLS cargo binding. Our results suggest that delivery of import receptors and NLS cargoes to nuclear pores may complement selective translocation through the pores as a functional mechanism for regulating transport of proteins into the nucleus.
    Full-text · Article · Jun 2009 · Biophysical Journal
  • [Show abstract] [Hide abstract] ABSTRACT: The universal structural role of collagen fiber networks has motivated the development of collagen gels, films, coatings, injectables, and other formulations. However, reported synthetic collagen fiber fabrication schemes have either culminated in short, discontinuous fiber segments at unsuitably low production rates, or have incompletely replicated the internal fibrillar structure that dictates fiber mechanical and biological properties. We report a continuous extrusion system with an off-line phosphate buffer incubation step for the manufacture of synthetic collagen fiber. Fiber with a cross-section of 53+ or - 14 by 21 + or - 3 microm and an ultimate tensile strength of 94 + or - 19 MPa was continuously produced at 60 m/hr from an ultrafiltered monomeric collagen solution. The effect of collagen solution concentration, flow rate, and spinneret size on fiber size was investigated. The fiber was further characterized by microdifferential scanning calorimetry, transmission electron microscopy (TEM), second harmonic generation (SHG) analysis, and in a subcutaneous murine implant model. Calorimetry demonstrated stabilization of the collagen triple helical structure, while TEM and SHG revealed a dense, axially aligned D-periodic fibril structure throughout the fiber cross-section. Implantation of glutaraldehyde crosslinked and noncrosslinked fiber in the subcutaneous tissue of mice demonstrated limited inflammatory response and biodegradation after a 6-week implant period.
    No preview · Article · Jan 2009 · Journal of Biomedical Materials Research Part B Applied Biomaterials

Publication Stats

2k Citations
137.86 Total Impact Points


  • 2005-2012
    • Emory University
      • Department of Physics
      Atlanta, Georgia, United States
  • 1998-2001
    • Massachusetts Institute of Technology
      • Department of Mechanical Engineering
      Cambridge, Massachusetts, United States
  • 1996-2001
    • University of Illinois, Urbana-Champaign
      • Department of Physics
      Urbana, Illinois, United States
  • 1997
    • University of North Carolina at Chapel Hill
      • Lineberger Comprehensive Cancer Center
      North Carolina, United States