Influence of Nonspecific Interactions on Protein Associations: Implications for Biochemistry In Vivo.

The cellular interior is composed of a variety of microenvironments defined by distinct local compositions and composition-dependent intermolecular interactions. We review the various types of nonspecific interactions between proteins and between proteins and other macromolecules and supramolecular structures that influence the state of association and functional properties of a given protein existing within a particular microenvironment at a particular point in time. The present state of knowledge is summarized, and suggestions for fruitful directions of research are offered.

Molecular Epigenetics: Chemical Biology Tools Come of Age.

The field of epigenetics has exploded over the last two decades, revealing an astonishing level of complexity in the way genetic information is stored and accessed in eukaryotes. This expansion of knowledge, which is very much ongoing, has been made possible by the availability of evermore sensitive and precise molecular tools. This review focuses on the increasingly important role that chemistry plays in this burgeoning field. In an effort to make these contributions more accessible to the nonspecialist, we group available chemical approaches into those that allow the covalent structure of the protein and DNA components of chromatin to be manipulated, those that allow the activity of myriad factors that act on chromatin to be controlled, and those that allow the covalent structure and folding of chromatin to be characterized. The application of these tools is illustrated through a series of case studies that highlight how the molecular precision afforded by chemistry is being used to establish causal biochemical relationships at the heart of epigenetic regulation.

From Bioorganic Models to Cells.

I endeavor to share how various choices-some deliberate, some unconscious-and the unmistakable influence of many others shaped my scientific pursuits. I am fascinated by how two long-term, major streams of my research, DNA replication and purine biosynthesis, have merged with unexpected interconnections. If I have imparted to many of the talented individuals who have passed through my lab a degree of my passion for uncloaking the mysteries hidden in scientific research and an understanding of the honesty and rigor it demands and its impact on the world community, then my mentorship has been successful.

Short- and Long-Term Adaptation to Altered Levels of Glucose: Fifty Years of Scientific Adventure.

My graduate and postdoctoral training in metabolism and enzymology eventually led me to study the short- and long-term regulation of glucose and lipid metabolism. In the early phase of my career, my trainees and I identified, purified, and characterized a variety of phosphofructokinase enzymes from mammalian tissues. These studies led us to discover fructose 2,6-P2, the most potent activator of phosphofructokinase and glycolysis. The discovery of fructose 2,6-P2 led to the identification and characterization of the tissue-specific bifunctional enzyme 6-phosphofructo-2-kinase:fructose 2,6-bisphosphatase. We discovered a glucose signaling mechanism by which the liver maintains glucose homeostasis by regulating the activities of this bifunctional enzyme. With a rise in glucose, a signaling metabolite, xylulose 5-phosphate, triggers rapid activation of a specific protein phosphatase (PP2ABδC), which dephosphorylates the bifunctional enzyme, thereby increasing fructose 2,6-P2 levels and upregulating glycolysis. These endeavors paved the way for us to initiate the later phase of my career in which we discovered a new transcription factor termed the carbohydrate response element binding protein (ChREBP). Now ChREBP is recognized as the masterregulator controlling conversion of excess carbohydrates to storage of fat in the liver. ChREBP functions as a central metabolic coordinator that responds to nutrients independently of insulin. The ChREBP transcription factor facilitates metabolic adaptation to excess glucose, leading to obesity and its associated diseases.

Optobiochemistry: Genetically Encoded Control of Protein Activity by Light.

Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light. Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques.

At the Intersection of Chemistry, Biology, and Medicine.

After an undergraduate degree in biology at Harvard, I started graduate school at The Rockefeller Institute for Medical Research in New York City in July 1965. I was attracted to the chemical side of biochemistry and joined Fritz Lipmann's large, hierarchical laboratory to study enzyme mechanisms. That work led to postdoctoral research with Robert Abeles at Brandeis, then a center of what, 30 years later, would be called chemical biology. I spent 15 years on the Massachusetts Institute of Technology faculty, in both the Chemistry and Biology Departments, and then 26 years on the Harvard Medical School Faculty. My research interests have been at the intersection of chemistry, biology, and medicine. One unanticipated major focus has been investigating the chemical logic and enzymatic machinery of natural product biosynthesis, including antibiotics and antitumor agents. In this postgenomic era it is now recognized that there may be from 105 to 106 biosynthetic gene clusters as yet uncharacterized for potential new therapeutic agents.

A Method for the Acute and Rapid Degradation of Endogenous Proteins.

Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.

Science as a Way of Knowing: From Protein Machines to Evidence-Based Decisions.

The 2016 Lasker∼Koshland Special Achievement Award will be presented to Bruce Alberts for a lifetime career of outstanding scientific discovery and inspiring leadership and mentorship in promoting fundamental research, science education, and rational, evidence-based values worldwide.

Veritas per structuram.

When I entered graduate school in 1963, the golden age of molecular biology had just begun, and myoglobin was the only protein with a known high-resolution structure. The romance of working out the structure of a virus by X-ray crystallography nonetheless captured both my imagination and the ensuing 15 years of my scientific life, during which "protein crystallography" began to morph into "structural biology." The course of the research recounted here follows the broader, 50-year trajectory of structural biology, as I could rarely resist opportunities to capitalize on new technologies when they opened some interesting part of biology to three-dimensional rigor. That fascination shows no sign of subsiding.

It seems like only yesterday.

I spent my childhood and adolescence in North and South Carolina, attended Duke University, and then entered Duke Medical School. One year in the laboratory of George Schwert in the biochemistry department kindled my interest in biochemistry. After one year of residency on the medical service of Duke Hospital, chaired by Eugene Stead, I joined the group of Arthur Kornberg at Stanford Medical School as a postdoctoral fellow. Two years later I accepted a faculty position at Harvard Medical School, where I remain today. During these 50 years, together with an outstanding group of students, postdoctoral fellows, and collaborators, I have pursued studies on DNA replication. I have experienced the excitement of discovering a number of important enzymes in DNA replication that, in turn, triggered an interest in the dynamics of a replisome. My associations with industry have been stimulating and fostered new friendships. I could not have chosen a better career.

Blended Learning Improves Science Education.

Blended learning is an emerging paradigm for science education but has not been rigorously assessed. We performed a randomized controlled trial of blended learning. We found that in-class problem solving improved exam performance, and video assignments increased attendance and satisfaction. This validates a new model for science communication and education.

Mapping Nucleosome Resolution Chromosome Folding in Yeast by Micro-C.

We describe a Hi-C-based method, Micro-C, in which micrococcal nuclease is used instead of restriction enzymes to fragment chromatin, enabling nucleosome resolution chromosome folding maps. Analysis of Micro-C maps for budding yeast reveals abundant self-associating domains similar to those reported in other species, but not previously observed in yeast. These structures, far shorter than topologically associating domains in mammals, typically encompass one to five genes in yeast. Strong boundaries between self-associating domains occur at promoters of highly transcribed genes and regions of rapid histone turnover that are typically bound by the RSC chromatin-remodeling complex. Investigation of chromosome folding in mutants confirms roles for RSC, "gene looping" factor Ssu72, Mediator, H3K56 acetyltransferase Rtt109, and the N-terminal tail of H4 in folding of the yeast genome. This approach provides detailed structural maps of a eukaryotic genome, and our findings provide insights into the machinery underlying chromosome compaction.

Activity-based profiling of proteases.

Proteolytic enzymes are key signaling molecules in both normal physiological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochemical methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the application of ABPs to protease enzymology, discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers.

Meet the authors: Grazia Pellicanò and Rodrigo Bermejo.

We talk to first and last authors of "Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse," Grazia Pellicanò and Rodrigo Bermejo, about their paper in this issue of Molecular Cell, the critical role of their collaborators, and the research in the Bermejo lab based in Madrid.

Foundations of biomolecular modeling.

The 2013 Nobel Prize in Chemistry has been awarded to Martin Karplus, Michael Levitt, and Arieh Warshel for "development of multiscale models for complex chemical systems." The honored work from the 1970s has provided a foundation for the widespread activities today in modeling organic and biomolecular systems.

Cultivating a Safe Space: An Interview with Dr. Donita Brady.

Dr. Brady shares her experiences in academia, the importance of a supportive environment, and what the scientific community can do to support underrepresented scientists. We hope her perspective encourages our readers to think about steps that can be taken to promote diversity and combat racial bias. The opinions expressed here are those of Dr. Brady and may not reflect the views of her institution.

The fires of life.

This retrospective recounts the hunt for the mechanism of mitochondrial ATP synthesis, the early days of research on mitochondrial formation, and some of the colorful personalities dominating these often dramatic and emotional efforts. The narrative is set against the backdrop of postwar Austria and Germany and the stream of young scientists who had to leave their countries to receive postdoctoral training abroad. Many of them--including the author--chose the laboratory of a scientist their country had expelled a few decades before. The article concludes with some thoughts on the uniqueness of U.S. research universities and a brief account of the struggles to revive science in Europe.

Measurements and implications of the membrane dipole potential.

There are three kinds of membrane potentials: the surface potentials, resulting from the accumulation of charges at the membrane surfaces; the transmembrane potential, determined by imbalance of charge in the aqueous solutions; and the dipole potential, a membrane-internal potential from the dipolar components of the phospholipids and interface water. The absolute value of the dipole potential has been very difficult to measure, although its value has been estimated to be in the range of 200-1,000 mV from ion translocation rates (determined by the planar lipid bilayer method), the surface potential of lipid monolayers (determined by the lipid monolayer method), molecular-dynamics calculations, and electron scattering using cryoelectron microscopy (cryo-EM). Spectroscopy methods have also been used to monitor the dipole potential changes on the basis of the observed fluorescence changes of voltage-sensitive probes. The dipole potential accounts for the much larger permeability of a bare phospholipid membrane to anions than cations and affects the conformation and function of membrane proteins.

An outreach activity to enhance biochemistry pedagogy.

Students are self-motivated to learn when provided opportunities that connect theory and real-world applications. Here, we describe for biochemistry majors a newborn screening-focused outreach activity that seeks to develop students' mastery of disciplinary content and soft skills (e.g., critical thinking, teamwork, effective communication, community engagement) and to enhance student engagement.

My life with nature.

After a childhood in Germany and being a youth in Grand Forks, North Dakota, I went to Harvard University, then to graduate school in biochemistry at the University of Wisconsin. Then to Washington University and Stanford University for postdoctoral training in biochemistry and genetics. Then at the University of Wisconsin, as a professor in the Department of Biochemistry and the Department of Genetics, I initiated research on bacterial chemotaxis. Here, I review this research by me and by many, many others up to the present moment. During the past few years, I have been studying chemotaxis and related behavior in animals, namely in Drosophila fruit flies, and some of these results are presented here. My current thinking is described.