Modern experimental kinetics of protein folding began in the early 1990s with the introduction of nanosecond laser pulses to trigger the folding reaction, providing an almost 10
-fold improvement in ...time resolution over the stopped-flow method being employed at the time. These experiments marked the beginning of the "fast-folding" subfield that enabled investigation of the kinetics of formation of secondary structural elements and disordered loops for the first time, as well as the fastest folding proteins. When I started to work on this subject, a fast folding protein was one that folded in milliseconds. There were, moreover, no analytical theoretical models and no atomistic or coarse-grained molecular dynamics simulations to describe the mechanism. Two of the most important discoveries from my lab since then are a protein that folds in hundreds of nanoseconds, as determined from nanosecond laser temperature experiments, and the discovery that the theoretically predicted barrier crossing time is about the same for proteins that differ in folding rates by 10
-fold, as determined from single molecule fluorescence measurements. We also developed what has been called the "Hückel model" of protein folding, which quantitatively explains a wide range of equilibrium and kinetic measurements. This retrospective traces the history of contributions to the "fast folding" subfield from my lab until about 3 years ago, when I left protein folding to spend the rest of my research career trying to discover an inexpensive drug for treating sickle cell disease.
Although the root cause of sickle cell disease is the polymerization of hemoglobin S (HbS) to form fibers that make red cells less flexible, most drugs currently being assessed in clinical trials are ...targeting the downstream sequelae of this primary event. Less attention has been devoted to investigation of the multiple ways in which fiber formation can be inhibited. In this article, we describe the molecular rationale for 5 distinct approaches to inhibiting polymerization and also discuss progress with the few antipolymerization drugs currently in clinical trials.
•Single molecule FRET experiments determine average transition path times.•Observed times are extremely close to those found in all-atom MD simulations.•Times are very similar for fast-folding and ...slow-folding proteins.•Longer times for designed protein are due to slower reaction coordinate diffusion.•Slower diffusion on reaction coordinate results from non-native interactions.
The transition path is the tiny segment of a single molecule trajectory when the free energy barrier between states is crossed and for protein folding contains all of the information about the self-assembly mechanism. As a first step toward obtaining structural information during the transition path from experiments, single molecule FRET spectroscopy has been used to determine average transition path times from a photon-by-photon analysis of fluorescence trajectories. These results, obtained for several different proteins, have already provided new and demanding tests that support both the accuracy of all-atom molecular dynamics simulations and the basic postulates of energy landscape theory of protein folding.
Kramers developed the theory on how chemical reaction rates are influenced by the viscosity of the medium. At the viscosity of water, the kinetics of unimolecular reactions are described by diffusion ...of a Brownian particle over a free-energy barrier separating reactants and products. For reactions in solution this famous theory extended Eyring's transition state theory, and is widely applied in physics, chemistry and biology, including to reactions as complex as protein folding. Because the diffusion coefficient of Kramers' theory is determined by the dynamics in the sparsely populated region of the barrier top, its properties have not been directly measured for any molecular system. Here we show that the Kramers diffusion coefficient and free-energy barrier can be characterized by measuring the temperature- and viscosity-dependence of the transition path time for protein folding. The transition path is the small fraction of an equilibrium trajectory for a single molecule when the free-energy barrier separating two states is actually crossed. Its duration, the transition path time, can now be determined from photon trajectories for single protein molecules undergoing folding/unfolding transitions. Our finding of a long transition path time with an unusually small solvent viscosity dependence suggests that internal friction as well as solvent friction determine the Kramers diffusion coefficient for α-helical proteins, as opposed to a breakdown of his theory, which occurs for many small-molecule reactions. It is noteworthy that the new and fundamental information concerning Kramers' theory and the dynamics of barrier crossings obtained here come from experiments on a protein rather than a much simpler chemical or physical system.
The recent availability of long equilibrium simulations of protein folding in atomistic detail for more than 10 proteins allows us to identify the key interactions driving folding. We find that the ...collective fraction of native amino acid contacts, Q , captures remarkably well the transition states for all the proteins with a folding free energy barrier. Going beyond this global picture, we devise two different measures to quantify the importance of individual interresidue contacts in the folding mechanism: (i) the log-ratio of lifetimes of contacts during folding transition paths and in the unfolded state and (ii) a Bayesian measure of how predictive the formation of each contact is for being on a transition path. Both of these measures indicate that native, or near-native, contacts are important for determining mechanism, as might be expected. More remarkably, however, we found that for almost all the proteins, with the designed protein α ₃D being a notable exception, nonnative contacts play no significant part in determining folding mechanisms.
Basic research on hemoglobin has been essential for understanding the origin and treatment of many hematological disorders due to abnormal hemoglobins. The most important of the hemoglobinopathies is ...sickle cell disease - Linus Pauling's “molecular disease” that gave birth to molecular medicine. In this review, I will describe the contributions of basic biophysical research on normal and sickle cell hemoglobin (HbS) to understanding the molecular pathogenesis of the disease and providing the conceptual basis for the various approaches to drug therapy that target HbS polymerization. Most prominent among these are the experimental results on the solubility of HbS as a function of oxygen saturation explained by the allosteric model of Monod, Wyman, and Changeux and the Gill-Wyman thermodynamic linkage relation between solubility and oxygen binding, the solubility of mixtures of HbS with normal or fetal hemoglobin explained by Minton's thermodynamic model, and the highly unusual kinetics of HbS polymerization explained by a novel double nucleation mechanism that also accounts for the aggregation kinetics of the Alzheimer's peptide. The HbS polymerization kinetics are of great importance to understanding the pathophysiology and clinical course, as well as guiding drug development for treating this common and severe disease. The article focuses primarily on experimental and theoretical results from my lab, so it is not a comprehensive review of the subject.
70 years ago, Linus Pauling, the legendary genius of 20th century chemistry, published his famous work on the molecular cause of sickle cell disease, a paper that gave birth to what is now called ...molecular medicine. In this paper, Pauling left important questions unanswered that have motivated an enormous amount of scientific and clinical research since then. This retrospective discusses the basic science studies that have answered those questions directly related to the kinetics and thermodynamics of hemoglobin S polymerization.
A complete understanding of a protein-folding mechanism requires description of the distribution of microscopic pathways that connect the folded and unfolded states. This distribution can, in ...principle, be described by computer simulations and theoretical models of protein folding, but is hidden in conventional experiments on large ensembles of molecules because only average properties are measured. A long-term goal of single-molecule fluorescence studies is to time-resolve the structural events as individual molecules make transitions between folded and unfolded states. Although such studies are still in their infancy, the work till now shows great promise and has already produced novel and important information on current issues in protein folding that has been impossible or difficult to obtain from ensemble measurements.
The transition path is the tiny fraction of an equilibrium molecular trajectory when a transition occurs as the free-energy barrier between two states is crossed. It is a single-molecule property ...that contains all the mechanistic information on how a process occurs. As a step toward observing transition paths in protein folding, we determined the average transition-path time for a fast-and a slow-folding protein from a photon-by-photon analysis of fluorescence trajectories in singlemolecule Förster resonance energy transfer experiments. Whereas the folding rate coefficients differ by a factor of 10,000, the transition-path times differ by a factor of less than 5, which shows that a fast-and a slow-folding protein take almost the same time to fold when folding actually happens. A very simple model based on energy landscape theory can explain this result.
Understanding the epidemiologic profile of the life course of mental disorders is fundamental for research and planning for health care. Although previous studies have used population surveys, ...informative and complementary estimates can be derived from population-based registers.
To derive comprehensive and precise estimates of the incidence rate of and lifetime risk for any mental disorder and a range of specific mental disorders.
We conducted a follow-up study of all Danish residents (5.6 million persons), to whom all treatment is provided by the government health care system without charge to the patient, from January 1, 2000, through December 31, 2012 (total follow-up, 59.5 million person-years). During the study period, 320,543 persons received first lifetime treatment in a psychiatric setting for any mental disorder; 489,006 persons were censored owing to death; and 69,987 persons were censored owing to emigration. Specific categories of mental disorders investigated included organic mental disorders, substance abuse disorders, schizophrenia, mood disorders, anxiety, eating disorders, personality disorders, mental retardation, pervasive developmental disorders, and behavioral and emotional disorders.
Age and sex.
Sex- and age-specific incidence rates and cumulative incidences and sex-specific lifetime risks.
During the course of life, 37.66% of females (95% CI, 37.52%-37.80%) and 32.05% of males (31.91%-32.19%) received their first treatment in a psychiatric setting for any mental disorder. The occurrence of mental disorders varied markedly between diagnostic categories and by sex and age. The sex- and age-specific incidence rates for many mental disorders had a single peak incidence rate during the second and third decades of life. Some disorders had a second peak in the sex- and age-specific incidence rate later in life.
This nationwide study provides a first comprehensive assessment of the lifetime risks for treated mental disorders. Approximately one-third of the Danish population received treatment for mental disorders. The distinct signatures of the different mental disorders with respect to sex and age have important implications for service planning and etiologic research.
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